Bio

Bio


My laboratory investigates the structure and function of biological systems using many tools and methods, always with a strong physical perspective. Four interconnected themes are being pursued.

First, we have a long-standing interest in the mechanism of light-driven, long-distance electron transfer in photosynthetic reaction centers, one of the fastest known reactions. This is being studied by femtosecond fluorescence and transient absorption spectroscopy, manipulation in electric fields, site-specific mutagenesis and some novel types of Stark spectroscopy we have developed and applied to many types of molecules.

Second, elated methods are also being used to probe excited state dynamics, solvation, and electronic structure in variants of green fluorescent protein (GFP), widely used in cell biology. Recently, methods have been developed that allow complete synthetic control of GFP so that any unnatural functionality can be introduced.

Third, we are broadly interested in electrostatics in proteins and how electrostatics affect function. Our current work uses vibrational probes whose sensitivity to electric fields can be calibrated by Stark spectroscopy. Probes are introduced on inhibitors, by modification of amino acids and by incorporation of unnatural amino acids, and these are used to map electric fields for comparison with simulations and as probes of the active site electrostatics in enzymes.

Fourth, we use supported lipid bilayers as mimics for cell surfaces and as tools in biotechnology. A broad vision is to engineer interfaces between hard surfaces and soft materials, ultimately leading to sophisticated biocompatible interfaces that can be used to control, interrogate or organize complex living systems. Recent work addresses the formation of domains and protein associations with these domains, interactions of DNA, proteins and cells with supported bilayers, and the mechanism of vesicle fusion. This work has motivated the development of advanced optical microscopy methods for probing the interface between membranes on solid supports and cell membranes, potentially with nm vertical resolution. A novel type of imaging mass spectrometry is being applied to characterize the lateral organization and composition of bilayers and associated membranes with 50 nm resolution.

Academic Appointments


Boards, Advisory Committees, Professional Organizations


  • Please see website, http://www.stanford.edu/group/boxer/ (2013 - Present)

Professional Education


  • Ph.D., Please see website, http://www.stanford.edu/group/boxer/

Research & Scholarship

Current Research and Scholarly Interests


Please visit my website for complete information:
http://www.stanford.edu/group/boxer/

Teaching

2013-14 Courses


Graduate and Fellowship Programs


Publications

Journal Articles


  • Individual Vesicle Fusion Events Mediated by Lipid-Anchored DNA BIOPHYSICAL JOURNAL van Lengerich, B., Rawle, R. J., Bendix, P. M., Boxer, S. G. 2013; 105 (2): 409-419

    Abstract

    Membrane fusion consists of a complex rearrangement of lipids and proteins that results in the merger of two lipid bilayers. We have developed a model system that employs synthetic DNA-lipid conjugates as a surrogate for the membrane proteins involved in the biological fusion reaction. We previously showed that complementary DNA-lipids, inserted into small unilamellar vesicles, can mediate membrane fusion in bulk. Here, we use a model membrane architecture developed in our lab to directly observe single-vesicle fusion events using fluorescence microscopy. In this system, a planar tethered membrane patch serves as the target membrane for incoming vesicles. This allows us to quantify the kinetics and characteristics of individual fusion events from the perspective of the lipids or the DNA-lipids involved in the process. We find that the fusion pathways are heterogeneous, with an arrested hemi-fusion state predominating, and we quantitate the outcome and rate of fusion events to construct a mechanistic model of DNA-mediated vesicle fusion. The waiting times between docking and fusion are distributed exponentially, suggesting that fusion occurs in a single step. Our analysis indicates that when two lipid bilayers are brought into close proximity, fusion occurs spontaneously, with little or no dependence on the number of DNA hybrids formed.

    View details for DOI 10.1016/j.bpj.2013.05.056

    View details for Web of Science ID 000321941700014

    View details for PubMedID 23870262

  • Quantitative dissection of hydrogen bond-mediated proton transfer in the ketosteroid isomerase active site PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sigala, P. A., Fafarman, A. T., Schwans, J. P., Fried, S. D., Fenn, T. D., Caaveiro, J. M., Pybus, B., Ringe, D., Petsko, G. A., Boxer, S. G., Herschlag, D. 2013; 110 (28): E2552-E2561

    Abstract

    Hydrogen bond networks are key elements of protein structure and function but have been challenging to study within the complex protein environment. We have carried out in-depth interrogations of the proton transfer equilibrium within a hydrogen bond network formed to bound phenols in the active site of ketosteroid isomerase. We systematically varied the proton affinity of the phenol using differing electron-withdrawing substituents and incorporated site-specific NMR and IR probes to quantitatively map the proton and charge rearrangements within the network that accompany incremental increases in phenol proton affinity. The observed ionization changes were accurately described by a simple equilibrium proton transfer model that strongly suggests the intrinsic proton affinity of one of the Tyr residues in the network, Tyr16, does not remain constant but rather systematically increases due to weakening of the phenol-Tyr16 anion hydrogen bond with increasing phenol proton affinity. Using vibrational Stark spectroscopy, we quantified the electrostatic field changes within the surrounding active site that accompany these rearrangements within the network. We were able to model these changes accurately using continuum electrostatic calculations, suggesting a high degree of conformational restriction within the protein matrix. Our study affords direct insight into the physical and energetic properties of a hydrogen bond network within a protein interior and provides an example of a highly controlled system with minimal conformational rearrangements in which the observed physical changes can be accurately modeled by theoretical calculations.

    View details for DOI 10.1073/pnas.1302191110

    View details for Web of Science ID 000321827000006

    View details for PubMedID 23798390

  • Colocalization of the Ganglioside G(M1) and Cholesterol Detected by Secondary Ion Mass Spectrometry JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Lozano, M. M., Liu, Z., Sunnick, E., Janshoff, A., Kumar, K., Boxer, S. G. 2013; 135 (15): 5620-5630

    Abstract

    The characterization of the lateral organization of components in biological membranes and the evolution of this arrangement in response to external triggers remain a major challenge. The concept of lipid rafts is widely invoked; however, direct evidence of the existence of these ephemeral entities remains elusive. We report here the use of secondary ion mass spectrometry (SIMS) to image the cholesterol-dependent cohesive phase separation of the ganglioside GM1 into nano- and microscale assemblies in a canonical lipid raft composition of lipids. This assembly of domains was interrogated in a model membrane system composed of palmitoyl sphingomyelin (PSM), cholesterol, and an unsaturated lipid (dioleoylphosphatidylcholine, DOPC). Orthogonal isotopic labeling of every lipid bilayer component and monofluorination of GM1 allowed generation of molecule specific images using a NanoSIMS. Simultaneous detection of six different ion species in SIMS, including secondary electrons, was used to generate ion ratio images whose signal intensity values could be correlated to composition through the use of calibration curves from standard samples. Images of this system provide the first direct, molecule specific, visual evidence for the colocalization of cholesterol and GM1 in supported lipid bilayers and further indicate the presence of three compositionally distinct phases: (1) the interdomain region; (2) micrometer-scale domains (d > 3 ?m); (3) nanometer-scale domains (d = 100 nm to 1 ?m) localized within the micrometer-scale domains and the interdomain region. PSM-rich, nanometer-scale domains prefer to partition within the more ordered, cholesterol-rich/DOPC-poor/GM1-rich micrometer-scale phase, while GM1-rich, nanometer-scale domains prefer to partition within the surrounding, disordered, cholesterol-poor/PSM-rich/DOPC-rich interdomain phase.

    View details for DOI 10.1021/ja310831m

    View details for Web of Science ID 000317872800026

    View details for PubMedID 23514537

  • Thermodynamic framework for identifying free energy inventories of enzyme catalytic cycles. Proceedings of the National Academy of Sciences of the United States of America Fried, S. D., Boxer, S. G. 2013; 110 (30): 12271-6

    Abstract

    Pauling's suggestion that enzymes are complementary in structure to the activated complexes of the reactions they catalyze has provided the conceptual basis to explain how enzymes obtain their fantastic catalytic prowess, and has served as a guiding principle in drug design for over 50 y. However, this model by itself fails to predict the magnitude of enzymes' rate accelerations. We construct a thermodynamic framework that begins with the classic concept of differential binding but invokes additional terms that are needed to account for subtle effects in the catalytic cycle's proton inventory. Although the model presented can be applied generally, this analysis focuses on ketosteroid isomerase (KSI) as an example, where recent experiments along with a large body of kinetic and thermodynamic data have provided strong support for the noncanonical thermodynamic contribution described. The resulting analysis precisely predicts the free energy barrier of KSI's reaction as determined from transition-state theory using only empirical thermodynamic data. This agreement is suggestive that a complete free energy inventory of the KSI catalytic cycle has been identified.

    View details for PubMedID 23840058

  • GFP Variants with Alternative ?-Strands and Their Application as Light-driven Protease Sensors: A Tale of Two Tails. Journal of the American Chemical Society Do, K., Boxer, S. G. 2013; 135 (28): 10226-9

    Abstract

    Green fluorescent protein (GFP) variants that carry one extra strand 10 (s10) were created and characterized, and their possible applications were explored. These proteins can fold with either one or the other s10, and the ratio of the two folded forms, unambiguously distinguished by their resulting colors, can be systematically modulated by mutating the residues on s10 or by changing the lengths of the two inserted linker sequences that connect each s10 to the rest of the protein. We have discovered robust empirical rules that accurately predict the product ratios of any given construct in both bacterial and mammalian expressions. Exploiting earlier studies on photodissociation of cut s10 from GFP (Do and Boxer, 2011), ratiometric protease sensors were designed from the construct by engineering a specific protease cleavage site into one of the inserted loops, where the bound s10 is replaced by the other strand upon protease cleavage and irradiation with light to switch its color. Since the conversion involves a large spectral shift, these genetically encoded sensors display a very high dynamic range. Further engineering of this class of proteins guided by mechanistic understanding of the light-driven process will enable interesting and useful application of the protein.

    View details for PubMedID 23819615

  • Experimental Quantification of Electrostatics in X-H center dot center dot center dot pi Hydrogen Bonds JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Saggu, M., Levinson, N. M., Boxer, S. G. 2012; 134 (46): 18986-18997

    Abstract

    Hydrogen bonds are ubiquitous in chemistry and biology. The physical forces that govern hydrogen-bonding interactions have been heavily debated, with much of the discussion focused on the relative contributions of electrostatic vs quantum mechanical effects. In principle, the vibrational Stark effect, the response of a vibrational mode to electric field, can provide an experimental method for parsing such interactions into their electrostatic and nonelectrostatic components. In a previous study we showed that, in the case of relatively weak O-H···π hydrogen bonds, the O-H bond displays a linear response to an electric field, and we exploited this response to demonstrate that the interactions are dominated by electrostatics (Saggu, M.; Levinson, N. M.; Boxer, S. G. J. Am. Chem. Soc.2011, 133, 17414-17419). Here we extend this work to other X-H···π interactions. We find that the response of the X-H vibrational probe to electric field appears to become increasingly nonlinear in the order O-H < N-H < S-H. The observed effects are consistent with differences in atomic polarizabilities of the X-H groups. Nonetheless, we find that the X-H stretching vibrations of the model compounds indole and thiophenol report quantitatively on the electric fields they experience when complexed with aromatic hydrogen-bond acceptors. These measurements can be used to estimate the electrostatic binding energies of the interactions, which are found to agree closely with the results of energy calculations. Taken together, these results highlight that with careful calibration vibrational probes can provide direct measurements of the electrostatic components of hydrogen bonds.

    View details for DOI 10.1021/ja305575t

    View details for Web of Science ID 000311324900023

  • Site-Specific Measurement of Water Dynamics in the Substrate Pocket of Ketosteroid Isomerase Using Time-Resolved Vibrational Spectroscopy JOURNAL OF PHYSICAL CHEMISTRY B Jha, S. K., Ji, M., Gaffney, K. J., Boxer, S. G. 2012; 116 (37): 11414-11421

    Abstract

    Little is known about the reorganization capacity of water molecules at the active sites of enzymes and how this couples to the catalytic reaction. Here, we study the dynamics of water molecules at the active site of a highly proficient enzyme, ?(5)-3-ketosteroid isomerase (KSI), during a light-activated mimic of its catalytic cycle. Photoexcitation of a nitrile-containing photoacid, coumarin183 (C183), mimics the change in charge density that occurs at the active site of KSI during the first step of the catalytic reaction. The nitrile of C183 is exposed to water when bound to the KSI active site, and we used time-resolved vibrational spectroscopy as a site-specific probe to study the solvation dynamics of water molecules in the vicinity of the nitrile. We observed that water molecules at the active site of KSI are highly rigid, during the light-activated catalytic cycle, compared to the solvation dynamics observed in bulk water. On the basis of this result, we hypothesize that rigid water dipoles at the active site might help in the maintenance of the preorganized electrostatic environment required for efficient catalysis. The results also demonstrate the utility of nitrile probes in measuring the dynamics of local (H-bonded) water molecules in contrast to the commonly used fluorescence methods which measure the average behavior of primary and subsequent spheres of solvation.

    View details for DOI 10.1021/jp305225r1

    View details for Web of Science ID 000308855800003

    View details for PubMedID 22931297

  • Solvent-Induced Infrared Frequency Shifts in Aromatic Nitriles Are Quantitatively Described by the Vibrational Stark Effect JOURNAL OF PHYSICAL CHEMISTRY B Levinson, N. M., Fried, S. D., Boxer, S. G. 2012; 116 (35): 10470-10476

    Abstract

    The physical properties of solvents strongly affect the spectra of dissolved solutes, and this phenomenon can be exploited to gain insight into the solvent-solute interaction. The large solvatochromic shifts observed for many dye molecules in polar solvents are due to variations in the solvent reaction field, and these shifts are widely used to estimate the change in the dye's dipole moment upon photoexcitation, which is typically on the order of ?1-10 D. In contrast, the change in dipole moment for vibrational transitions is approximately 2 orders of magnitude smaller. Nonetheless, vibrational chromophores display significant solvatochromism, and the relative contributions of specific chemical interactions and electrostatic interactions are debated, complicating the interpretation of vibrational frequency shifts in complex systems such as proteins. Here we present a series of substituted benzonitriles that display widely varying degrees of vibrational solvatochromism. In most cases, this variation can be quantitatively described by the experimentally determined Stark tuning rate, coupled with a simple Onsager-like model of solvation, reinforcing the view that vibrational frequency shifts are largely caused by electrostatic interactions. In addition, we discuss specific cases where continuum solvation models fail to predict solvatochromic shifts, revealing the necessity for more advanced theoretical models that capture local aspects of solute-solvent interactions.

    View details for DOI 10.1021/jp301054e

    View details for Web of Science ID 000308339400007

    View details for PubMedID 22448878

  • Photochemistry of a Bacterial Photosynthetic Reaction Center Missing the Initial Bacteriochlorophyll Electron Acceptor JOURNAL OF PHYSICAL CHEMISTRY B Carter, B., Boxer, S. G., Holten, D., Kirmaier, C. 2012; 116 (33): 9971-9982

    Abstract

    A novel chromophore composition of the bacterial photosynthetic reaction center (RC) has been discovered: RCs lacking the L-side monomeric bacteriochlorophyll chromophore result from mutation of the native isoleucine at M204 to glutamine in Rhodobacter capsulatus . This conclusion is obtained from 77 K UV-vis spectroscopy and pigment extractions of the I(M204)Q mutant and seven variants containing the I(M204)Q plus other mutations. The oxidation potential of the primary electron donor P (a dimer of bacteriochlorophylls) was measured for three of the mutants and found to be 50-65 mV lower than in wild-type RCs. Ultrafast transient absorption measurements reveal (minimally) two subpopulations of P* that have distinct lifetimes and photochemical outcomes for all mutants containing I(M204)Q. In one subpopulation P* decays solely by internal conversion to the ground state. In the other subpopulation P* decays by electron transfer to the normally inactive M-side bacteriopheophytin (H(M)) in competition with internal conversion to the ground state. When a Tyr residue is substituted for the native Phe at L181 near the M-side monomeric bacteriochlorophyll (B(M)), the rate of electron transfer to H(M) is increased about 4-fold.

    View details for DOI 10.1021/jp305276m

    View details for Web of Science ID 000307749100011

    View details for PubMedID 22876929

  • A Solvatochromic Model Calibrates Nitriles' Vibrational Frequencies to Electrostatic Fields JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Bagchi, S., Fried, S. D., Boxer, S. G. 2012; 134 (25): 10373-10376

    Abstract

    Electrostatic interactions provide a primary connection between a protein's three-dimensional structure and its function. Infrared probes are useful because vibrational frequencies of certain chemical groups, such as nitriles, are linearly sensitive to local electrostatic field and can serve as a molecular electric field meter. IR spectroscopy has been used to study electrostatic changes or fluctuations in proteins, but measured peak frequencies have not been previously mapped to total electric fields, because of the absence of a field-frequency calibration and the complication of local chemical effects such as H-bonds. We report a solvatochromic model that provides a means to assess the H-bonding status of aromatic nitrile vibrational probes and calibrates their vibrational frequencies to electrostatic field. The analysis involves correlations between the nitrile's IR frequency and its (13)C chemical shift, whose observation is facilitated by a robust method for introducing isotopes into aromatic nitriles. The method is tested on the model protein ribonuclease S (RNase S) containing a labeled p-CN-Phe near the active site. Comparison of the measurements in RNase S against solvatochromic data gives an estimate of the average total electrostatic field at this location. The value determined agrees quantitatively with molecular dynamics simulations, suggesting broader potential for the use of IR probes in the study of protein electrostatics.

    View details for DOI 10.1021/ja303895k

    View details for Web of Science ID 000305716700017

    View details for PubMedID 22694663

  • Structural and Spectroscopic Analysis of the Kinase Inhibitor Bosutinib and an Isomer of Bosutinib Binding to the Abl Tyrosine Kinase Domain PLOS ONE Levinson, N. M., Boxer, S. G. 2012; 7 (4)

    Abstract

    Chronic myeloid leukemia (CML) is caused by the kinase activity of the BCR-Abl fusion protein. The Abl inhibitors imatinib, nilotinib and dasatinib are currently used to treat CML, but resistance to these inhibitors is a significant clinical problem. The kinase inhibitor bosutinib has shown efficacy in clinical trials for imatinib-resistant CML, but its binding mode is unknown. We present the 2.4 Å structure of bosutinib bound to the kinase domain of Abl, which explains the inhibitor's activity against several imatinib-resistant mutants, and reveals that similar inhibitors that lack a nitrile moiety could be effective against the common T315I mutant. We also report that two distinct chemical compounds are currently being sold under the name "bosutinib", and report spectroscopic and structural characterizations of both. We show that the fluorescence properties of these compounds allow inhibitor binding to be measured quantitatively, and that the infrared absorption of the nitrile group reveals a different electrostatic environment in the conserved ATP-binding sites of Abl and Src kinases. Exploiting such differences could lead to inhibitors with improved selectivity.

    View details for DOI 10.1371/journal.pone.0029828

    View details for Web of Science ID 000305012700002

    View details for PubMedID 22493660

  • Ribonuclease S Dynamics Measured Using a Nitrile Label with 2D IR Vibrational Echo Spectroscopy JOURNAL OF PHYSICAL CHEMISTRY B Bagchi, S., Boxer, S. G., Fayer, M. D. 2012; 116 (13): 4034-4042

    Abstract

    A nitrile-labeled amino acid, p-cyanophenylalanine, is introduced near the active site of the semisynthetic enzyme ribonuclease S to serve as a probe of protein dynamics and fluctuations. Ribonuclease S is the limited proteolysis product of subtilisin acting on ribonuclease A, and consists of a small fragment including amino acids 1-20, the S-peptide, and a larger fragment including residues 21-124, the S-protein. A series of two-dimensional vibrational echo experiments performed on the nitrile-labeled S-peptide and the RNase S are described. The time-dependent changes in the two-dimensional infrared vibrational echo line shapes are analyzed using the center line slope method to obtain the frequency-frequency correlation function (FFCF). The observations show that the nitrile probe in the S-peptide has dynamics that are similar to, but faster than, those of the single amino acid p-cyanophenylalanine in water. In contrast, the dynamics of the nitrile label when the peptide is bound to form ribonuclease S are dominated by homogeneous dephasing (motionally narrowed) contributions with only a small contribution from very fast inhomogeneous structural dynamics. The results provide insights into the nature of the structural dynamics of the ribonuclease S complex. The equilibrium dynamics of the nitrile labeled S-peptide and the ribonuclease S complex are also investigated by molecular dynamics simulations. The experimentally determined FFCFs are compared to the FFCFs obtained from the molecular dynamics simulations, thereby testing the capacity of simulations to determine the amplitudes and time scales of protein structural fluctuations on fast time scales under thermal equilibrium conditions.

    View details for DOI 10.1021/jp2122856

    View details for Web of Science ID 000302337000015

    View details for PubMedID 22417088

  • Quantitative, directional measurement of electric field heterogeneity in the active site of ketosteroid isomerase PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Fafarman, A. T., Sigala, P. A., Schwans, J. P., Fenn, T. D., Herschlag, D., Boxer, S. G. 2012; 109 (6): E299-E308

    Abstract

    Understanding the electrostatic forces and features within highly heterogeneous, anisotropic, and chemically complex enzyme active sites and their connection to biological catalysis remains a longstanding challenge, in part due to the paucity of incisive experimental probes of electrostatic properties within proteins. To quantitatively assess the landscape of electrostatic fields at discrete locations and orientations within an enzyme active site, we have incorporated site-specific thiocyanate vibrational probes into multiple positions within bacterial ketosteroid isomerase. A battery of X-ray crystallographic, vibrational Stark spectroscopy, and NMR studies revealed electrostatic field heterogeneity of 8 MV/cm between active site probe locations and widely differing sensitivities of discrete probes to common electrostatic perturbations from mutation, ligand binding, and pH changes. Electrostatic calculations based on active site ionization states assigned by literature precedent and computational pK(a) prediction were unable to quantitatively account for the observed vibrational band shifts. However, electrostatic models of the D40N mutant gave qualitative agreement with the observed vibrational effects when an unusual ionization of an active site tyrosine with a pK(a) near 7 was included. UV-absorbance and (13)C NMR experiments confirmed the presence of a tyrosinate in the active site, in agreement with electrostatic models. This work provides the most direct measure of the heterogeneous and anisotropic nature of the electrostatic environment within an enzyme active site, and these measurements provide incisive benchmarks for further developing accurate computational models and a foundation for future tests of electrostatics in enzymatic catalysis.

    View details for DOI 10.1073/pnas.1111566109

    View details for Web of Science ID 000299925000004

    View details for PubMedID 22308339

  • Evaluation of the Energetics of the Concerted Acid-Base Mechanism in Enzymatic Catalysis: The Case of Ketosteroid Isomerase JOURNAL OF PHYSICAL CHEMISTRY B Fried, S. D., Boxer, S. G. 2012; 116 (1): 690-697

    Abstract

    Structures of enzymes invariably reveal the proximity of acidic and basic residues to reactive sites on the substrate, so it is natural and common to suggest that enzymes employ concerted mechanisms to catalyze their difficult reactions. Ketosteroid isomerase (KSI) has served as a paradigm of enzymatic proton transfer chemistry, and its catalytic effect has previously been attributed to concerted proton transfer. We employ a specific inhibitor that contains an IR probe that reports directly and quantitatively on the ionization state of the ligand when bound in the active site of KSI. Measurement of the fractional ionization provides a missing link in a thermodynamic cycle that can discriminate the free energy advantage of a concerted versus nonconcerted mechanism. It is found that the maximum thermodynamic advantage that KSI could capture from a concerted mechanism (??G° = 0.5 kcal mol(-1)) is quite small.

    View details for DOI 10.1021/jp210544w

    View details for Web of Science ID 000298978100081

    View details for PubMedID 22148842

  • Thermodynamics, Kinetics, and Photochemistry of beta-Strand Association and Dissociation in a Split-GFP System JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Do, K., Boxer, S. G. 2011; 133 (45): 18078-18081

    Abstract

    Truncated green fluorescent protein (GFP) that is refolded after removing the 10th ?-strand can readily bind to a synthetic strand to recover the absorbance and fluorescence of the whole protein. This allows rigorous experimental determination of thermodynamic and kinetic parameters of the split system including the equilibrium constant and the association/dissociation rates, which enables residue-specific analysis of peptide-protein interactions. The dissociation rate of the noncovalently bound strand is observed by strand exchange that is accompanied by a color change, and surprisingly, the rate is greatly enhanced by light irradiation. This peptide-protein photodissociation is a very unusual phenomenon and can potentially be useful for introducing spatially and temporally well-defined perturbations to biological systems as a genetically encoded caged protein.

    View details for DOI 10.1021/ja207985w

    View details for Web of Science ID 000297381200020

    View details for PubMedID 21981121

  • Direct Measurements of Electric Fields in Weak OH-pi Hydrogen Bonds JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Saggu, M., Levinson, N. M., Boxer, S. G. 2011; 133 (43): 17414-17419

    Abstract

    Hydrogen bonds and aromatic interactions are of widespread importance in chemistry, biology, and materials science. Electrostatics play a fundamental role in these interactions, but the magnitude of the electric fields that support them has not been quantified experimentally. Phenol forms a weak hydrogen bond complex with the ?-cloud of benzene, and we used this as a model system to study the role of electric fields in weak OH···? hydrogen bonds. The effects of complex formation on the vibrational frequency of the phenol OH or OD stretches were measured in a series of benzene-based aromatic solvents. Large shifts are observed and these can be converted into electric fields via the measured vibrational Stark effect. A comparison of the measured fields with quantum chemical calculations demonstrates that calculations performed in the gas phase are surprisingly effective at capturing the electrostatics observed in solution. The results provide quantitative measurements of the magnitude of electric fields and electrostatic binding energies in these interactions and suggest that electrostatics dominate them. The combination of vibrational Stark effect (VSE) measurements of electric fields and high-level quantum chemistry calculations is a general strategy for quantifying and characterizing the origins of intermolecular interactions.

    View details for DOI 10.1021/ja2069592

    View details for Web of Science ID 000297380900046

    View details for PubMedID 21936553

  • Vesicle Fusion Observed by Content Transfer across a Tethered Lipid Bilayer BIOPHYSICAL JOURNAL Rawle, R. J., van Lengerich, B., Chung, M., Bendix, P. M., Boxer, S. G. 2011; 101 (8): L37-L39

    Abstract

    Synaptic transmission is achieved by exocytosis of small, synaptic vesicles containing neurotransmitters across the plasma membrane. Here, we use a DNA-tethered freestanding bilayer as a target architecture that allows observation of content transfer of individual vesicles across the tethered planar bilayer. Tethering and fusion are mediated by hybridization of complementary DNA-lipid conjugates inserted into the two membranes, and content transfer is monitored by the dequenching of an aqueous content dye. By analyzing the diffusion profile of the aqueous dye after vesicle fusion, we are able to distinguish content transfer across the tethered bilayer patch from vesicle leakage above the patch.

    View details for DOI 10.1016/j.bpj.2011.09.023

    View details for Web of Science ID 000296075800001

    View details for PubMedID 22004762

  • Electrostatic Fields near the Active Site of Human Aldose Reductase: 2. New Inhibitors and Complications Caused by Hydrogen Bonds BIOCHEMISTRY Xu, L., Cohen, A. E., Boxer, S. G. 2011; 50 (39): 8311-8322

    Abstract

    Vibrational Stark effect spectroscopy was used to measure electrostatic fields in the hydrophobic region of the active site of human aldose reductase (hALR2). A new nitrile-containing inhibitor was designed and synthesized, and the X-ray structure of its complex, along with cofactor NADP(+), with wild-type hALR2 was determined at 1.3 Å resolution. The nitrile is found to be in the proximity of T113, consistent with a hydrogen bond interaction. Two vibrational absorption peaks were observed at room temperature in the nitrile region when the inhibitor binds to wild-type hALR2, indicating that the nitrile probe experiences two different microenvironments, and these could be empirically separated into a hydrogen-bonded and non-hydrogen-bonded population by comparison with the T113A mutant, in which a hydrogen bond to the nitrile is not present. Classical molecular dynamics simulations based on the structure predict a double-peak distribution in protein electric fields projected along the nitrile probe. The interpretation of these two peaks as a hydrogen bond formation-dissociation process between the probe nitrile group and a nearby amino acid side chain is used to explain the observation of two IR bands, and the simulations were used to investigate the molecular details of this conformational change. Hydrogen bonding complicates the simplest analysis of vibrational frequency shifts as being due solely to electrostatic interactions through the vibrational Stark effect, and the consequences of this complication are discussed.

    View details for DOI 10.1021/bi200930f

    View details for Web of Science ID 000295187200007

    View details for PubMedID 21859105

  • Direct measurement of the protein response to an electrostatic perturbation that mimics the catalytic cycle in ketosteroid isomerase PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Jha, S. K., Ji, M., Gaffney, K. J., Boxer, S. G. 2011; 108 (40): 16612-16617

    Abstract

    Understanding how electric fields and their fluctuations in the active site of enzymes affect efficient catalysis represents a critical objective of biochemical research. We have directly measured the dynamics of the electric field in the active site of a highly proficient enzyme, ?(5)-3-ketosteroid isomerase (KSI), in response to a sudden electrostatic perturbation that simulates the charge displacement that occurs along the KSI catalytic reaction coordinate. Photoexcitation of a fluorescent analog (coumarin 183) of the reaction intermediate mimics the change in charge distribution that occurs between the reactant and intermediate state in the steroid substrate of KSI. We measured the electrostatic response and angular dynamics of four probe dipoles in the enzyme active site by monitoring the time-resolved changes in the vibrational absorbance (IR) spectrum of a spectator thiocyanate moiety (a quantitative sensor of changes in electric field) placed at four different locations in and around the active site, using polarization-dependent transient vibrational Stark spectroscopy. The four different dipoles in the active site remain immobile and do not align to the changes in the substrate electric field. These results indicate that the active site of KSI is preorganized with respect to functionally relevant changes in electric fields.

    View details for DOI 10.1073/pnas.1113874108

    View details for Web of Science ID 000295536000031

    View details for PubMedID 21949360

  • Phosphate Vibrations Probe Local Electric Fields and Hydration in Biomolecules JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Levinson, N. M., Bolte, E. E., Miller, C. S., Corcelli, S. A., Boxer, S. G. 2011; 133 (34): 13236-13239

    Abstract

    The role of electric fields in important biological processes such as binding and catalysis has been studied almost exclusively by computational methods. Experimental measurements of the local electric field in macromolecules are possible using suitably calibrated vibrational probes. Here we demonstrate that the vibrational transitions of phosphate groups are highly sensitive to an electric field and show how that sensitivity can be quantified, allowing electric field measurements to be made in phosphate-containing biological systems without chemical modification.

    View details for DOI 10.1021/ja2042589

    View details for Web of Science ID 000295551600008

    View details for PubMedID 21809829

  • Stability of DNA-Tethered Lipid Membranes with Mobile Tethers LANGMUIR Chung, M., Boxer, S. G. 2011; 27 (9): 5492-5497

    Abstract

    We recently introduced two approaches for tethering planar lipid bilayers as membrane patches to either a supported lipid bilayer or DNA-functionalized surface using DNA hybridization (Chung, M.; Lowe, R. D.; Chan, Y-H. M.; Ganesan, P. V.; Boxer, S. G. J. Struct. Biol.2009, 168, 190-9). When mobile DNA tethers are used, the tethered bilayer patches become unstable, while they are stable if the tethers are fixed on the surface. Because the mobile tethers between a patch and a supported lipid bilayer offer a particularly interesting architecture for studying the dynamics of membrane-membrane interactions, we have investigated the sources of instability, focusing on membrane composition. The most stable patches were made with a mixture of saturated lipids and cholesterol, suggesting an important role for membrane stiffness. Other factors such as the effect of tether length, lateral mobility, and patch membrane edge were also investigated. On the basis of these results, a model for the mechanism of patch destruction is developed.

    View details for DOI 10.1021/la200234h

    View details for Web of Science ID 000289742500040

    View details for PubMedID 21452847

  • Light-Activated Reassembly of Split Green Fluorescent Protein JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Kent, K. P., Boxer, S. G. 2011; 133 (11): 4046-4052

    Abstract

    Truncated green fluorescent protein (GFP) with the 11th ?-strand removed is potentially interesting for bioconjugation, imaging, and the preparation of semisynthetic proteins with novel spectroscopic or functional properties. Surprisingly, the truncated GFP generated by removing the 11th strand, once refolded, does not reassemble with a synthetic peptide corresponding to strand 11 but does reassemble following light activation. The mechanism of this process has been studied in detail by absorption, fluorescence, and Raman spectroscopy. The chromophore in this refolded truncated GFP is found to be in the trans configuration. Upon exposure to light a photostationary state is formed between the trans and cis conformations of the chromophore, and only truncated GFP with the cis configuration of the chromophore binds the peptide. A kinetic model describing the light-activated reassembly of this split GFP is discussed. This unique light-driven reassembly is potentially useful for controlling protein-protein interactions.

    View details for DOI 10.1021/ja110256c

    View details for Web of Science ID 000288889900064

    View details for PubMedID 21351768

  • Nitrile Bonds as Infrared Probes of Electrostatics in Ribonuclease S JOURNAL OF PHYSICAL CHEMISTRY B Fafarman, A. T., Boxer, S. G. 2010; 114 (42): 13536-13544

    Abstract

    Three different nitrile-containing amino acids, p-cyanophenylalanine, m-cyanophenylalanine, and S-cyanohomocysteine, have been introduced near the active site of the semisynthetic enzyme ribonuclease S (RNase S) to serve as probes of electrostatic fields. Vibrational Stark spectra, measured directly on the probe-modified proteins, confirm the predominance of the linear Stark tuning rate in describing the sensitivity of the nitrile stretch to external electric fields, a necessary property for interpreting observed frequency shifts as a quantitative measure of local electric fields that can be compared with simulations. The X-ray structures of these nitrile-modified RNase variants and enzymatic assays demonstrate minimal perturbation to the structure and function, respectively, by the probes and provide a context for understanding the influence of the environment on the nitrile stretching frequency. We examine the ability of simulation techniques to recapitulate the spectroscopic properties of these nitriles as a means to directly test a computational electrostatic model for proteins, specifically that in the ubiquitous Amber-99 force field. Although qualitative agreement between theory and experiment is observed for the largest shifts, substantial discrepancies are observed in some cases, highlighting the ongoing need for experimental metrics to inform the development of theoretical models of electrostatic fields in proteins.

    View details for DOI 10.1021/jp106406p

    View details for Web of Science ID 000283110500033

    View details for PubMedID 20883003

  • Decomposition of Vibrational Shifts of Nitriles into Electrostatic and Hydrogen-Bonding Effects JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Fafarman, A. T., Sigala, P. A., Herschlag, D., Boxer, S. G. 2010; 132 (37): 12811-12813

    Abstract

    Infrared (IR) band shifts of isolated vibrational transitions can serve as quantitative and directional probes of local electrostatic fields, due to the vibrational Stark effect. However, departures from the Stark model can arise when the probe participates in specific, chemical interactions, such as direct hydrogen bonding. We present a method to identify and correct for these departures based on comparison of (13)C NMR chemical shifts and IR frequencies each calibrated in turn by a solvatochromic model. We demonstrate how the tandem use of these experimental observables can be applied to a thiocyanate-modified protein, ketosteroid isomerase, and show, by comparison to structural models, that changes in electrostatic field can be measured within the complex protein environment even in the background of direct hydrogen bonding to the probe.

    View details for DOI 10.1021/ja104573b

    View details for Web of Science ID 000282013700018

    View details for PubMedID 20806897

  • Covalent Attachment of Lipid Vesicles to a Fluid-Supported Bilayer Allows Observation of DNA-Mediated Vesicle Interactions LANGMUIR van Lengerich, B., Rawle, R. J., Boxer, S. G. 2010; 26 (11): 8666-8672

    Abstract

    Specific membrane interactions such as lipid vesicle docking and fusion can be mediated by synthetic DNA-lipid conjugates as a model for the protein-driven processes that are ubiquitous in biological systems. Here we present a method of tethering vesicles to a supported lipid bilayer that allows the simultaneous deposition of cognate vesicle partners displaying complementary DNA, resulting in well-mixed populations of tethered vesicles that are laterally mobile. Vesicles are covalently attached to a supporting lipid bilayer using a DNA-templated click reaction; then DNA-mediated interactions between tethered vesicles are triggered by spiking the salt concentration. These interactions, such as docking and fusion, can then be observed for individual vesicles as they collide on the surface. The architecture of this new system also permits control over the number of lipid anchors that tether the vesicle to the supporting bilayer. The diffusion coefficient of tethered vesicles anchored by two lipids is approximately 1.6-fold slower than that of vesicles anchored only with a single lipid, consistent with a simple physical model.

    View details for DOI 10.1021/la904822f

    View details for Web of Science ID 000277928100134

    View details for PubMedID 20180548

  • Solvation Response along the Reaction Coordinate in the Active Site of Ketosteroid Isomerase JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Childs, W., Boxer, S. G. 2010; 132 (18): 6474-6480

    Abstract

    A light-activated reaction analog has been developed to mimic the catalytic reaction cycle of Delta(5)-3-ketosteroid isomerase to probe the functionally relevant protein solvation response to the catalytic charge transfer. Delta(5)-3-ketosteroid isomerase from Pseudomonas putida catalyzes a C-H bond cleavage and formation through an enolate intermediate. Conversion of the ketone substrate to the enolate intermediate is simulated by a photoacid bound to the active site oxyanion hole. In the ground state, the photoacid electrostatically resembles the enolate intermediate while the low pK(a) excited state resembles the ketone starting material. Time-resolved fluorescence experiments with photoacids coumarin 183 and equilenin show the active site of Delta(5)-3-ketosteroid isomerase to be largely unperturbed by the light-activated reaction. The small solvation response for the photoacid at the active site as compared with a simple solvent suggests the active site does not significantly change its electrostatic environment during the catalytic cycle. Instead, the reaction takes place in an electrostatically preorganized environment.

    View details for DOI 10.1021/ja1007849

    View details for Web of Science ID 000277445400038

    View details for PubMedID 20397697

  • Proton Affinity of the Oxyanion Hole in the Active Site of Ketosteroid Isomerase BIOCHEMISTRY Childs, W., Boxer, S. G. 2010; 49 (12): 2725-2731

    Abstract

    The absorption spectra of a series of inhibitors bound at the active site of Delta(5)-3-ketosteroid isomerase from Pseudomonas putida were found to exhibit substantial variations in the contributions of the protonated and deprotonated forms. Systematic variation of the inhibitor solution pK(a) combined with a method of quantifying the contributions of each protonation state showed the oxyanion hole in the active site of wild-type Delta(5)-3-ketosteroid isomerase to have a proton affinity equal to a solution pK(a) of 10.05 +/- 0.03, which is similar to the measured pK(a) (10.0) of the reaction intermediate. This observation supports the prediction of Cleland, Kreevoy, Frey, Gassman, and Gerlt that an enzyme utilizing a strong hydrogen bond for catalysis matches the proton affinity of the protein to the intermediate [Cleland, W. W., and Kreevoy, M. M. (1994) Science 264, 1887-1890; Frey, P. A., Whitt, S., and Tobin, J. (1994) Science 264, 1927-1930; Gerlt, J. A., and Gassman, P. G. (1993) Biochemistry 32, 11934-11952]. As the difference in proton affinity decreases, the strength of the hydrogen bond increases, and the closely matched proton affinity between the active site and the reaction intermediate supports the possibility that a short, strong hydrogen bond is catalytically relevant in Delta(5)-3-ketosteroid isomerase.

    View details for DOI 10.1021/bi100074s

    View details for Web of Science ID 000275858400012

    View details for PubMedID 20143849

  • Synthetic Control of Green Fluorescent Protein JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Kent, K. P., Oltrogge, L. M., Boxer, S. G. 2009; 131 (44): 15988-?

    Abstract

    Semisynthetic green fluorescent proteins (GFPs) can be prepared by producing truncated GFPs recombinantly and assembling them with synthetic beta-strands of GFP. The yield from expressing the truncated GFPs is low, and the chromophore is either partially formed or not formed. An alternative method is presented in which full-length proteins are produced recombinantly with a protease site inserted between the structural element to be removed and the rest of the protein. The native peptide can then be replaced by cutting the protease site with trypsin, denaturing in guanidine hydrochloride to disrupt the complex, separating the native peptide from the rest of the protein by size exclusion, and refolding the protein in the presence of a synthetic peptide. We show that this method allows for removal and replacement of the interior chromophore containing helix and that the GFP barrel is capable of inducing chromophore formation in a synthetic interior helix.

    View details for DOI 10.1021/ja906303f

    View details for Web of Science ID 000271513700012

    View details for PubMedID 19839621

  • DNA-tethered membranes formed by giant vesicle rupture JOURNAL OF STRUCTURAL BIOLOGY Chung, M., Lowe, R. D., Chan, Y. M., Ganesan, P. V., Boxer, S. G. 2009; 168 (1): 190-199

    Abstract

    We have developed a strategy for preparing tethered lipid bilayer membrane patches on solid surfaces by DNA hybridization. In this way, the tethered membrane patch is held at a controllable distance from the surface by varying the length of the DNA used. Two basic strategies are described. In the first, single-stranded DNA strands are immobilized by click chemistry to a silica surface, whose remaining surface is passivated to prevent direct assembly of a solid supported bilayer. Then giant unilamellar vesicles (GUVs) displaying the antisense strand, using a DNA-lipid conjugate developed in earlier work [Chan, Y.-H.M., van Lengerich, B., et al., 2008. Lipid-anchored DNA mediates vesicle fusion as observed by lipid and content mixing. Biointerphases 3 (2), FA17-FA21], are allowed to tether, spread and rupture to form tethered bilayer patches. In the second, a supported lipid bilayer displaying DNA using the DNA-lipid conjugate is first assembled on the surface. Then GUVs displaying the antisense strand are allowed to tether, spread and rupture to form tethered bilayer patches. The essential difference between these methods is that the tethering hybrid DNA is immobile in the first, while it is mobile in the second. Both strategies are successful; however, with mobile DNA hybrids as tethers, the patches are unstable, while in the first strategy stable patches can be formed. In the case of mobile tethers, if different length DNA hybrids are present, lateral segregation by length occurs and can be visualized by fluorescence interference contrast microscopy making this an interesting model for interactions that occur in cell junctions. In both cases, lipid mobility is high and there is a negligible immobile fraction. Thus, these architectures offer a flexible platform for the assembly of lipid bilayers at a well-defined distance from a solid support.

    View details for DOI 10.1016/j.jsb.2009.06.015

    View details for Web of Science ID 000274799800021

    View details for PubMedID 19560541

  • A membrane interferometer PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ganesan, P. V., Boxer, S. G. 2009; 106 (14): 5627-5632

    Abstract

    Freestanding phospholipid bilayers have been assembled spanning shallow, micrometer-sized wells etched into a Si wafer substrate so that the bilayers are near (within hundreds of nanometers) but not in contact with the wafer surface. The proximity of the bilayers to the highly reflective Si/SiO(2) interface allows them to be probed by using fluorescence-interference techniques. These interferometry measurements show that the bilayers are curved and that the curvature can be varied by changes in osmotic pressure. Furthermore, the ionophore gramicidin can be incorporated into the bilayers, making them selectively permeable to monovalent cations. This freestanding architecture may overcome surface-interaction problems that occur when cell membrane proteins are introduced into solid supported bilayers, while also allowing for high-precision measurements of changes in fluorophore position by interferometry.

    View details for DOI 10.1073/pnas.0901770106

    View details for Web of Science ID 000264967500033

    View details for PubMedID 19307575

  • Trapping the P+BL- Initial Intermediate State of Charge Separation in Photosynthetic Reaction Centers from Rhodobacter capsulatus BIOCHEMISTRY Carter, B., Boxer, S. G., Holten, D., Kirmaier, C. 2009; 48 (12): 2571-2573

    Abstract

    The short-lived (<1 ps) initial intermediate state P(+)B(L)(-) in the photoinduced charge separation process of the bacterial photosynthetic reaction center has been trapped in two D(LL)-based Rhodobacter capsulatus mutants that have Tyr at position M208 and lack the bacteriopheophytin electron acceptor H(L). Transient state P(+)B(L)(-) is characterized by a 1017 nm bacteriochlorophyll anion absorption band and decays by charge recombination with a lifetime of several hundred picoseconds at 295 K. P(+)B(L)(-) is not observed in an otherwise identical mutant that has Phe at M208, which appears to make the state thermodynamically inaccessible from the excited primary electron donor P*.

    View details for DOI 10.1021/bi900282p

    View details for Web of Science ID 000264536500001

    View details for PubMedID 19245209

  • Stark Realities JOURNAL OF PHYSICAL CHEMISTRY B Boxer, S. G. 2009; 113 (10): 2972-2983

    Abstract

    Electric fields affect any process or transition that involves the movement of charge. Stark spectroscopy is a general term describing the study of spectral changes in the presence of electric fields, and it has proven to be a broadly useful approach for characterizing the change in dipole moment and polarizability for electronic and vibrational transitions. This article focuses primarily on the evolution of the approach and interconnected applications in diverse fields from our laboratory and prospects for the future. Our work began with studies of chromophores in photosynthetic reaction centers whose function is light-driven charge separation, so perturbations by an electric field were a natural approach. The same methods have been applied to many other biological and nonbiological chromophores. A common theme has been understanding the mechanism(s) of symmetry breaking in molecules or organized assemblies of high symmetry. Spectral shifts in organized systems due to mutations, conformational changes, and ligand binding can, in some cases, be interpreted as Stark shifts. In this case, Stark spectroscopy in a well-defined electric field provides a calibration of the probe transition's sensitivity to an electric field, and the Stark shifts of suitable probes can be used to measure the magnitude and direction of electric fields in proteins, nucleic acids, and membranes. Electric fields can also perturb the populations or reaction dynamics of processes where charge separation occurs. When detected by spectroscopic methods, we call these nonclassical Stark effects. Nonclassical Stark effects arise in the spectroscopy of intervalence charge transfer transitions and both ground- and excited-state electron transfer reactions. Because the movement of charge is ubiquitous in chemistry, biology, and materials science and because electric fields directly affect the energetics of charge-separated species, many phenomena can be viewed as generalizations of the Stark effect.

    View details for DOI 10.1021/jp8067393

    View details for Web of Science ID 000263974900003

    View details for PubMedID 19708160

  • Effects of linker sequences on vesicle fusion mediated by lipid-anchored DNA oligonucleotides PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chan, Y. M., van Lengerich, B., Boxer, S. G. 2009; 106 (4): 979-984

    Abstract

    Synthetic lipid-oligonucleotide conjugates inserted into lipid vesicles mediate fusion when one population of vesicles displays the 5'-coupled conjugate and the other the 3'-coupled conjugate, so that anti-parallel hybridization allows the membrane surfaces to come into close proximity. Improved assays show that lipid mixing proceeds more quickly and to a much greater extent than content mixing, suggesting the latter is rate limiting. To test the effect of membrane-membrane spacing on fusion, a series of conjugates was constructed by adding 2-24 noncomplementary bases at the membrane-proximal ends of two complementary sequences. Increasing linker lengths generally resulted in progressively reduced rates and extents of lipid and content mixing, in contrast to higher vesicle docking rates. The relatively flexible, single-stranded DNA linker facilitates docking but allows greater spacing between the vesicles after docking, thus making the transition into fusion less probable, but not preventing it altogether. These experiments demonstrate the utility of DNA as a model system for fusion proteins, where sequence can easily be modified to systematically probe the effect of distance between bilayers in the fusion reaction.

    View details for DOI 10.1073/pnas.0812356106

    View details for Web of Science ID 000262831600006

    View details for PubMedID 19164559

  • Advances in Imaging Secondary Ion Mass Spectrometry for Biological Samples ANNUAL REVIEW OF BIOPHYSICS Boxer, S. G., Kraft, M. L., Weber, P. K. 2009; 38: 53-74

    Abstract

    Imaging mass spectrometry combines the power of mass spectrometry to identify complex molecules based on mass with sample imaging. Recent advances in secondary ion mass spectrometry have improved sensitivity and spatial resolution, so that these methods have the potential to bridge between high-resolution structures obtained by X-ray crystallography and cyro-electron microscopy and ultrastructure visualized by conventional light microscopy. Following background information on the method and instrumentation, we address the key issue of sample preparation. Because mass spectrometry is performed in high vacuum, it is essential to preserve the lateral organization of the sample while removing bulk water, and this has been a major barrier for applications to biological systems. Recent applications of imaging mass spectrometry to cell biology, microbial communities, and biosynthetic pathways are summarized briefly, and studies of biological membrane organization are described in greater depth.

    View details for DOI 10.1146/annurev.biophys.050708.133634

    View details for Web of Science ID 000268072100003

    View details for PubMedID 19086820

  • Charge Transfer in Photoacids Observed by Stark Spectroscopy JOURNAL OF PHYSICAL CHEMISTRY A Silverman, L. N., Spry, D. B., Boxer, S. G., Fayer, M. D. 2008; 112 (41): 10244-10249

    Abstract

    The charge redistribution upon photoexcitation is investigated for a series of pyrene photoacids to better understand the driving force behind excited-state proton-transfer processes. The changes in electric dipole for the lowest two electronic transitions ( (1)L b and (1)L a) are measured by Stark spectroscopy, and the magnitudes of charge transfer of the protonated and deprotonated states are compared. For neutral photoacids studied here, the results show that the amount of charge transfer depends more upon the electronic state that is excited than the protonation state. Transitions from the ground state to the (1)L b state result in a much smaller change in electric dipole than transitions to the (1)L a state. Conversely, for the cationic (ammonium) photoacid studied, photoexcitation of a particular electronic state results in much smaller charge transfer for the protonated state than for the deprotonated state.

    View details for DOI 10.1021/jp805189u

    View details for Web of Science ID 000259943100022

    View details for PubMedID 18798602

  • Deconstructing green fluorescent protein JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Kent, K. P., Childs, W., Boxer, S. G. 2008; 130 (30): 9664-?

    Abstract

    Green fluorescent protein (GFP) has been reassembled from two pieces, a large fragment 214 amino acids in length that is produced recombinantly (GFP 1-10) and a short synthetic peptide corresponding to the 11th stave of the beta-barrel that is 16 amino acids long (synthetic GFP 11), following a system developed by Waldo and co-workers (Cabantous, S.; et al. Nat. Biotechnol. 2005, 23, 102-7) as an in vivo probe for protein association and folding. We demonstrate that the reassembled protein has identical absorption and excited-state proton transfer dynamics as a whole protein of the identical sequence. We show that the reassembled protein can be taken apart and the peptide replaced with a different synthetic peptide designed to perturb the chromophore absorption. Thus, this semisynthetic reassembly process offers a general route for studying the assembly of the beta-barrel as well as the introduction of unnatural amino acids.

    View details for DOI 10.1021/ja803782x

    View details for Web of Science ID 000257902500022

    View details for PubMedID 18597452

  • Temperature dependence of electron transfer to the M-side bacteriopheophytin in Rhodobacter capsulatus reaction Centers JOURNAL OF PHYSICAL CHEMISTRY B Chuang, J. I., Boxer, S. G., Holten, D., Kirmaier, C. 2008; 112 (17): 5487-5499

    Abstract

    Subpicosecond time-resolved absorption measurements at 77 K on two reaction center (RC) mutants of Rhodobacter capsulatus are reported. In the D(LL) mutant the D helix of the M subunit has been substituted with the D helix from the L subunit, and in the D(LL)-FY(L)F(M) mutant, three additional mutations are incorporated that facilitate electron transfer to the M side of the RC. In both cases the helix swap has been shown to yield isolated RCs that are devoid of the native bacteriopheophytin electron carrier HL (Chuang, J. I.; Boxer, S. G.; Holten, D.; Kirmaier, C. Biochemistry 2006, 45, 3845-3851). For D(LL), depending whether the detergent Deriphat 160-C or N-lauryl-N,N-dimethylamine-N-oxide (LDAO) is used to suspend the RCs, the excited state of the primary electron donor (P*) decays to the ground state with an average lifetime at 77 K of 330 or 170 ps, respectively; however, in both cases the time constant obtained from single-exponential fits varies markedly as a function of the probe wavelength. These findings on the D(LL) RC are most easily explained in terms of a heterogeneous population of RCs. Similarly, the complex results for D(LL)-FY(L)F(M) in Deriphat-glycerol glass at 77 K are most simply explained using a model that involves (minimally) two distinct populations of RCs with very different photochemistry. Within this framework, in 50% of the D(LL)-FY(L)F(M) RCs in Deriphat-glycerol glass at 77 K, P* deactivates to the ground state with a time constant of approximately 400 ps, similar to the deactivation of P* in the D(LL) mutant at 77 K. In the other 50% of D(LL)-FY(L)F(M) RCs, P* has a 35 ps lifetime and decays via electron transfer to the M branch, giving P+HM- in high yield (> or =80%). This result indicates that P* --> P(+)H(M)(-) is roughly a factor of 2 faster at 77 K than at 295 K. In alternative homogeneous models the rate of this M-side electron-transfer process is the same or up to 2-fold slower at low temperature. A 2-fold increase in rate with a reduction in temperature is the same behavior found for the overall L-side process P* --> P(+)H(L)(-) in wild-type RCs. Our results suggest that, as for electron transfer on the L side, the M-side electron-transfer reaction P* --> P(+)H(M)(-) is an activationless process.

    View details for DOI 10.1021/jp800082m

    View details for Web of Science ID 000255292300031

    View details for PubMedID 18402487

  • Electrostatic fields near the active site of human aldose reductase: 1. New inhibitors and vibrational stark effect measurements BIOCHEMISTRY Webb, L. J., Boxer, S. G. 2008; 47 (6): 1588-1598

    Abstract

    Vibrational Stark effect spectroscopy was used to measure electrostatic fields in the hydrophobic region of the active site of human aldose reductase (hALR2). A new hALR2 inhibitor was designed and synthesized that contains a nitrile probe with a Stark tuning rate of 0.77 cm-1/(MV/cm). Mutations to amino acid residues in the vicinity of the nitrile functional group were selected based on electrostatics calculations, possible complications from hydrogen bonds near the nitrile, and comparison with the active site of human aldehyde reductase, whose structure is very similar. Changes in the absorption energy of the nitrile probe when bound to those mutated proteins were then used to quantify perturbations to the protein's electrostatic field. Electrostatic field changes as large as -10 MV/cm were observed. Measured electrostatic fields were compared to predictions based on continuum electrostatics calculations, revealing that substantial modifications to the calculation strategy are necessary. The effects of hydrogen bonding of amino acid side chains to the nitrile probe are considered, and applications of vibrational Stark effect spectroscopy to investigations of ligand binding and biological function are discussed.

    View details for DOI 10.1021/bi701708u

    View details for Web of Science ID 000252940600013

    View details for PubMedID 18205401

  • Stark spectroscopy of mixed-valence systems PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY A-MATHEMATICAL PHYSICAL AND ENGINEERING SCIENCES Silverman, L. N., Kanchanawong, P., Treynor, T. P., Boxer, S. G. 2008; 366 (1862): 33-45

    Abstract

    Many mixed-valence systems involve two or more states with different electric dipole moments whose magnitudes depend upon the charge transfer distance and the degree of delocalization; these systems can be interconverted by excitation of an intervalence charge transfer transition. Stark spectroscopy involves the interaction between the change in dipole moment of a transition and an electric field, so the Stark spectra of mixed-valence systems are expected to provide quantitative information on the degree of delocalization. In limiting cases, a classical Stark analysis can be used, but in intermediate cases the analysis is much more complex because the field affects not only the band position but also the intrinsic bandshape. Such non-classical Stark effects lead to widely different bandshapes. Several examples of both classes are discussed. Because electric fields are applied to immobilized samples, complications arise from inhomogeneous broadening, along with other effects that limit our ability to extract unique parameters in some cases. In the case of the radical cation of the special pair in photosynthetic reaction centres, where the mixed-valence system is in a very complex but structurally well-defined environment, a detailed analysis can be performed.

    View details for DOI 10.1098/rsta.2007.2137

    View details for Web of Science ID 000251716300004

    View details for PubMedID 17827128

  • Dynamic Stokes shift in green fluorescent protein variants PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Abbyad, P., Childs, W., Shi, X., Boxer, S. G. 2007; 104 (51): 20189-20194

    Abstract

    Solvent reorganization around the excited state of a chromophore leads to an emission shift to longer wavelengths during the excited-state lifetime. This solvation response is absent in wild-type green fluorescent protein, and this has been attributed to rigidity in the chromophore's environment necessary to exclude nonradiative transitions to the ground state. The fluorescent protein mPlum was developed via directed evolution by selection for red emission, and we use time-resolved fluorescence to study the dynamic Stokes shift through its evolutionary history. The far-red emission of mPlum is attributed to a picosecond solvation response that is observed at all temperatures above the glass transition. This time-dependent shift in emission is not observed in its evolutionary ancestors, suggesting that selective pressure has produced a chromophore environment that allows solvent reorganization. The evolutionary pathway and structures of related fluorescent proteins suggest the role of a single residue in close proximity to the chromophore as the primary cause of the solvation response.

    View details for DOI 10.1073/pnas.0706185104

    View details for Web of Science ID 000251885000009

    View details for PubMedID 18077381

  • Anomalous negative fluorescence Anisotropy in yellow fluorescent protein (YFP 10C): Quantitative analysis of FRET in YFP dimers BIOCHEMISTRY Shi, X., Basran, J., Seward, H. E., Childs, W., Bagshaw, C. R., Boxer, S. G. 2007; 46 (50): 14403-14417

    Abstract

    Yellow fluorescent protein (YFP) is widely used as a genetically encoded fluorescent marker in biology. In the course of a comprehensive study of this protein, we observed an unusual, negative fluorescence anisotropy at pH 6.0 (McAnaney, T. B., Zeng, W., Doe, C. F. E., Bhanji, N., Wakelin, S., Pearson, D. S., Abbyad, P., Shi, X., Boxer, S. G., and Bagshaw, C. R. (2005) Biochemistry 44, 5510-5524). Here we report that the fluorescence anisotropy of YFP 10C depends on protein concentration in the low micromolar range that was not expected. We propose that the negative anisotropy is a result of unidirectional Förster resonance energy transfer (FRET) in a dimer of YFP, with the donor chromophore in the neutral form and the acceptor chromophore in the anionic form. This unusual mechanism is supported by studies of a monomeric YFP (A206K YFP) and transient-absorption spectroscopy of YFP 10C. A detailed analysis of the chromophore transition dipole moment direction is presented. The anisotropy and rate constant of this energy transfer are consistent with values produced by an analysis of the dimer structure observed in crystals.

    View details for DOI 10.1021/bi701575n

    View details for Web of Science ID 000251547700008

    View details for PubMedID 18027983

  • Model membrane systems and their applications CURRENT OPINION IN CHEMICAL BIOLOGY Chan, Y. M., Boxer, S. G. 2007; 11 (6): 581-587

    Abstract

    The complexity of biological membranes has motivated the development of a wide variety of simpler model systems whose size, geometry, and composition can be tailored with great precision. Approaches highlighted in this review are illustrated in Figure 1 including vesicles, supported bilayers, and hybrid membrane systems. These have been used to study problems ranging from phase behavior to membrane fusion. Experimental membrane models continue to advance in complexity with respect to architecture, size, and composition, as do computer simulations of their properties and dynamics. Analytical techniques such as imaging secondary ion mass spectrometry have also been developed and refined to give increasing spatial resolution and information content on membrane composition and dynamics.

    View details for DOI 10.1016/j.cbpa.2007.09.020

    View details for Web of Science ID 000251954100002

    View details for PubMedID 17976391

  • Kinetics of DNA-mediated docking reactions between vesicles tethered to supported lipid bilayers PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chan, Y. M., Lenz, P., Boxer, S. G. 2007; 104 (48): 18913-18918

    Abstract

    Membrane-membrane recognition and binding are crucial in many biological processes. We report an approach to studying the dynamics of such reactions by using DNA-tethered vesicles as a general scaffold for displaying membrane components. This system was used to characterize the docking reaction between two populations of tethered vesicles that display complementary DNA. Deposition of vesicles onto a supported lipid bilayer was performed by using a microfluidic device to prevent mixing of the vesicles in bulk during sample preparation. Once tethered onto the surface, vesicles mixed via two-dimensional diffusion. DNA-mediated docking of two reacting vesicles results in their colocalization after collision and their subsequent tandem motion. Individual docking events and population kinetics were observed via epifluorescence microscopy. A lattice-diffusion simulation was implemented to extract from experimental data the probability, P(dock), that a collision leads to docking. For individual vesicles displaying small numbers of docking DNA, P(dock) shows a first-order relationship with copy number as well as a strong dependence on the DNA sequence. Both trends are explained by a model that includes both tethered vesicle diffusion on the supported bilayer and docking DNA diffusion over each vesicle's surface. These results provide the basis for the application of tethered vesicles to study other membrane reactions including protein-mediated docking and fusion.

    View details for DOI 10.1073/pnas0.706114104

    View details for Web of Science ID 000251498700010

    View details for PubMedID 18025472

  • Ultrafast excited-state dynamics in the green fluorescent protein variant S65T/H148D. 1. Mutagenesis and structural studies BIOCHEMISTRY Shu, X., Kallio, K., Shi, X., Abbyad, P., Kanchanawong, P., Childs, W., Boxer, S. G., Remington, S. J. 2007; 46 (43): 12005-12013

    Abstract

    Wild type green fluorescent protein (wt-GFP) and the variant S65T/H148D each exhibit two absorption bands, A and B, which are associated with the protonated and deprotonated chromophores, respectively. Excitation of either band leads to green emission. In wt-GFP, excitation of band A ( approximately 395 nm) leads to green emission with a rise time of 10-15 ps, due to excited-state proton transfer (ESPT) from the chromophore hydroxyl group to an acceptor. This process produces an anionic excited-state intermediate I* that subsequently emits a green photon. In the variant S65T/H148D, the A band absorbance maximum is red-shifted to approximately 415 nm, and as detailed in the accompanying papers, when the A band is excited, green fluorescence appears with a rise time shorter than the instrument time resolution ( approximately 170 fs). On the basis of the steady-state spectroscopy and high-resolution crystal structures of several variants described herein, it is proposed that in S65T/H148D, the red shift of absorption band A and the ultrafast appearance of green fluorescence upon excitation of band A are due to a very short (

    View details for DOI 10.1021/bi7009037

    View details for Web of Science ID 000250379900003

    View details for PubMedID 17918959

  • Ultrafast excited-state dynamics in the green fluorescent protein variant S65T/H148D. 2. Unusual photophysical properties BIOCHEMISTRY Shi, X., Abbyad, P., Shu, X., Kallio, K., Kanchanawong, P., Childs, W., Remington, S. J., Boxer, S. G. 2007; 46 (43): 12014-12025

    Abstract

    In the preceding accompanying paper [Shu, X., et al. (2007) Biochemistry 46, 12005-12013], the 1.5 A resolution crystal structure of green fluorescent protein (GFP) variant S65T/H148D is presented, and the possible consequences of an unusual short hydrogen bond (

    View details for DOI 10.1021/bi700904a

    View details for Web of Science ID 000250379900004

    View details for PubMedID 17918960

  • Vibrational stark effect probes for nucleic acids JOURNAL OF PHYSICAL CHEMISTRY B Silverman, L. N., Pitzer, M. E., Ankomah, P. O., Boxer, S. G., Fenlon, E. E. 2007; 111 (40): 11611-11613

    Abstract

    The vibrational Stark effect (VSE) has proven to be an effective method for the study of electric fields in proteins via the use of infrared probes. To explore the use of VSE in nucleic acids, we investigated the Stark spectroscopy of nine structurally diverse nucleosides. These nucleosides contained nitrile or azide probes in positions that correspond to both the major and minor grooves of DNA. The nitrile probes showed better characteristics and exhibited absorption frequencies over a broad range; that is, from 2253 cm-1 for 2'-O-cyanoethyl ribonucleosides 8 and 9 to 2102 cm(-1) for a 13C-labeled 5-thiocyanatomethyl-2'-deoxyuridine 3c. The largest Stark tuning rate observed was |Deltamu| = 1.1 cm(-1)/(MV/cm) for both 5-cyano-2'-deoxyuridine 1 and N2-nitrile-2'-deoxyguanosine 7. The latter is a particularly attractive probe because of its high extinction coefficient (epsilon = 412 M-1cm-1) and ease of incorporation into oligomers.

    View details for DOI 10.1021/jp0750912

    View details for Web of Science ID 000249986900001

    View details for PubMedID 17877390

  • Do ligand binding and solvent exclusion alter the electrostatic character within the oxyanion hole of an enzymatic active site? JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Sigala, P. A., Fafarman, A. T., Bogard, P. E., Boxer, S. G., Herschlag, D. 2007; 129 (40): 12104-?

    View details for DOI 10.1021/ja075605a

    View details for Web of Science ID 000249949600025

    View details for PubMedID 17854190

  • Measurement of solvation responses at multiple sites in a globular protein JOURNAL OF PHYSICAL CHEMISTRY B Abbyad, P., Shi, X., Childs, W., McAnaney, T. B., Cohen, B. E., Boxer, S. G. 2007; 111 (28): 8269-8276

    Abstract

    Proteins respond to electrostatic perturbations through complex reorganizations of their charged and polar groups, as well as those of the surrounding media. These solvation responses occur both in the protein interior and on its surface, though the exact mechanisms of solvation are not well understood, in part because of limited data on the solvation responses for any given protein. Here, we characterize the solvation kinetics at sites throughout the sequence of a small globular protein, the B1 domain of streptococcal protein G (GB1), using the synthetic fluorescent amino acid Aladan. Aladan was incorporated into seven different GB1 sites, and the time-dependent Stokes shift was measured over the femtosecond to nanosecond time scales by fluorescence upconversion and time-correlated single photon counting. The seven sites range from buried within the protein core to fully solvent-exposed on the protein surface, and are located on different protein secondary structures including beta-sheets, helices, and loops. The dynamics in the protein sites were compared against the free fluorophore in buffer. All protein sites exhibited an initial, ultrafast Stokes shift on the subpicosecond time scale similar to that observed for the free fluorophore, but smaller in magnitude. As the probe is moved from the surface to more buried sites, the dynamics of the solvation response become slower, while no clear correlation between dynamics and secondary structure is observed. We suggest that restricted movements of the surrounding protein residues give rise to the observed long time dynamics and that such movements comprise a large portion of the protein's solvation response. The proper treatment of dynamic Stokes shift data when the time scale for solvation is comparable to the fluorescence lifetime is discussed.

    View details for DOI 10.1021/jp0709104

    View details for Web of Science ID 000247966300045

    View details for PubMedID 17592867

  • Frictional drag and electrical manipulation of recombinant proteins in polymer-supported membranes LANGMUIR Tanaka, M., Hermann, J., Haase, I., Fischer, M., Boxer, S. G. 2007; 23 (10): 5638-5644

    Abstract

    We establish a lipid monolayer supported by a polymer interface that offers advantages over conventional solid-supported membranes for determining the frictional drag at the membrane-protein interface as well as for electric field manipulation of membrane-anchored proteins. Polymer-supported monolayers with functional lipid anchors allow for the specific docking of His-tagged green fluorescent protein variants (His-EGFP and His-DsRed tetramer) onto the membrane surface at a defined surface density. In the first part, we measure the lateral diffusion coefficients of lipids and proteins and calculate the frictional drag at the protein-membrane interface. The second part deals with the electric field-induced accumulation of recombinant proteins on a patterned surface. The mean drift velocity of proteins, which can be obtained analytically from the shape of the steady-state concentration gradient, can be controlled by tuning the interplay of electrophoresis and electroosmosis. The results demonstrate the potential of such molecular constructs for the local functionalization of solid substrates with membrane-associated proteins.

    View details for DOI 10.1021/la0628219

    View details for Web of Science ID 000246071800062

    View details for PubMedID 17408291

  • Site-specific conversion of cysteine thiols into thiocyanate creates an IR probe for electric fields in proteins JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Fafarman, A. T., Webb, L. J., Chuang, J. I., Boxer, S. G. 2006; 128 (41): 13356-13357

    Abstract

    The nitrile stretching mode of the thiocyanate moiety is a nearly ideal probe for measuring the local electric field arising from the organized environment of the interior of a protein. Nitriles were introduced into three proteins: ribonuclease S (RNase S), human aldose reductase (hALR2), and the reaction center (RC) of Rhodobacter capsulatus, through a facile synthetic scheme for the transformation of cysteine residues into thiocyanatoalanine. Vibrational Stark effect spectroscopy and Fourier transform infrared spectroscopy on the modified proteins demonstrated that thiocyanate residues are a highly general tool for probing electrostatic fields in proteins.

    View details for DOI 10.1021/ja0650403

    View details for Web of Science ID 000241157600020

    View details for PubMedID 17031938

  • Phase separation of lipid membranes analyzed with high-resolution secondary ion mass spectrometry SCIENCE Kraft, M. L., Weber, P. K., Longo, M. L., Hutcheon, I. D., Boxer, S. G. 2006; 313 (5795): 1948-1951

    Abstract

    Lateral variations in membrane composition are postulated to play a central role in many cellular events, but it has been difficult to probe membrane composition and organization on length scales of tens to hundreds of nanometers. We present a high-resolution imaging secondary ion mass spectrometry technique to reveal the lipid distribution within a phase-separated membrane with a lateral resolution of approximately 100 nanometers. Quantitative information about the chemical composition within small lipid domains was obtained with the use of isotopic labels to identify each molecular species. Composition variations were detected within some domains.

    View details for DOI 10.1126/science.1130279

    View details for Web of Science ID 000240832200047

    View details for PubMedID 17008528

  • Charge delocalization in the special-pair radical cation of mutant reaction centers of Rhodobacter sphaeroides from stark spectra and nonadiabatic spectral simulations JOURNAL OF PHYSICAL CHEMISTRY B Kanchanawong, P., Dahlbom, M. G., Treynor, T. P., Reimers, J. R., Hush, N. S., Boxer, S. G. 2006; 110 (37): 18688-18702

    Abstract

    Stark and absorption spectra for the hole-transfer band of the bacteriochlorophyll special pair in the wild-type and L131LH, M160LH, and L131LH/M160LH mutants of the bacterial reaction center of Rhodobacter sphaeroides are presented, along with extensive analyses based on nonadiabatic spectral simulations. Dramatic changes in the Stark spectra are induced by the mutations, changes that are readily interpreted in terms of the redox-energy asymmetry and degree of charge localization in the special-pair radical cation. The effect of mutagenesis on key properties such as the electronic coupling within the special pair and the reorganization energy associated with intervalence hole transfer are determined for the first time. Results for the L131LH and M160LH/L131LH mutants indicate that these species can be considered as influencing the special pair primarily through modulation of the redox asymmetry, as is usually conceptualized, but M160LH is shown to develop a wide range of effects that can be interpreted in terms of significant mutation-induced structural changes in and around the special pair. The nonadiabatic spectra simulations are performed using both a simple two-state 1-mode and an extensive four-state 70-mode model, which includes the descriptions of additional electronic states and explicitly treats the major vibrational modes involved. Excellent agreement between the two simulation approaches is obtained. The simple model is shown to reproduce key features of the Stark effect of the main intervalence transition, while the extensive model quantitatively reproduces most features of the observed spectra for both the electronic and the phase-phonon regions, thus giving a more comprehensive description of the effect of the mutations on the properties of the special-pair radical cation. These results for a series of closely related mixed-valence complexes show that the Stark spectra provide a sensitive indicator for the properties of the mixed-valence complexes and should serve as an instructive example on the application of nonadiabatic simulations to the study of mixed-valence complexes in general as well as other chemical systems akin to the photosynthetic special pair.

    View details for DOI 10.1021/jp0623894

    View details for Web of Science ID 000240496500089

    View details for PubMedID 16970500

  • Antibody evolution constrains conformational heterogeneity by tailoring protein dynamics PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Zimmermann, J., Oakman, E. L., Thorpe, I. F., Shi, X., Abbyad, P., Brooks, C. L., Boxer, S. G., Romesberg, F. E. 2006; 103 (37): 13722-13727

    Abstract

    The evolution of proteins with novel function is thought to start from precursor proteins that are conformationally heterogeneous. The corresponding genes may be duplicated and then mutated to select and optimize a specific conformation. However, testing this idea has been difficult because of the challenge of quantifying protein flexibility and conformational heterogeneity as a function of evolution. Here, we report the characterization of protein heterogeneity and dynamics as a function of evolution for the antifluorescein antibody 4-4-20. Using nonlinear laser spectroscopy, surface plasmon resonance, and molecular dynamics simulations, we demonstrate that evolution localized the Ab-combining site from a heterogeneous ensemble of conformations to a single conformation by introducing mutations that act cooperatively and over significant distances to rigidify the protein. This study demonstrates how protein dynamics may be tailored by evolution and has important implications for our understanding of how novel protein functions are evolved.

    View details for DOI 10.1073/pnas.0603282103

    View details for Web of Science ID 000240648300032

    View details for PubMedID 16954202

  • Electric fields at the active site of an enzyme: Direct comparison of experiment with theory SCIENCE Suydam, I. T., Snow, C. D., Pande, V. S., Boxer, S. G. 2006; 313 (5784): 200-204

    Abstract

    The electric fields produced in folded proteins influence nearly every aspect of protein function. We present a vibrational spectroscopy technique that measures changes in electric field at a specific site of a protein as shifts in frequency (Stark shifts) of a calibrated nitrile vibration. A nitrile-containing inhibitor is used to deliver a unique probe vibration to the active site of human aldose reductase, and the response of the nitrile stretch frequency is measured for a series of mutations in the enzyme active site. These shifts yield quantitative information on electric fields that can be directly compared with electrostatics calculations. We show that extensive molecular dynamics simulations and ensemble averaging are required to reproduce the observed changes in field.

    View details for DOI 10.1126/science.1127159

    View details for Web of Science ID 000239008000037

    View details for PubMedID 16840693

  • Diffusive dynamics of vesicles tethered to a fluid supported bilayer by single-particle tracking LANGMUIR Yoshina-Ishii, C., Chan, Y. M., Johnson, J. M., Kung, L. A., Lenz, P., Boxer, S. G. 2006; 22 (13): 5682-5689

    Abstract

    We recently introduced a method to tether intact phospholipid vesicles onto a fluid supported lipid bilayer using DNA hybridization (Yoshina-Ishii, C.; Miller, G. P.; Kraft, M. L; Kool, E. T.; Boxer, S. G. J. Am. Chem. Soc. 2005, 127, 1356-1357). Once tethered, the vesicles can diffuse in two dimensions parallel to the supported membrane surface. The average diffusion coefficient, D, is typically 0.2 microm(2)/s; this is 3-5 times smaller than for individual lipid or DNA-lipid conjugate diffusion in supported bilayers. In this article, we investigate the origin of this difference in the diffusive dynamics of tethered vesicles by single-particle tracking under collision-free conditions. D is insensitive to tethered vesicle size from 30 to 200 nm, as well as a 3-fold change in the viscosity of the bulk medium. The addition of macromolecules such as poly(ethylene glycol) reversibly stops the motion of tethered vesicles without causing the exchange of lipids between the tethered vesicle and supported bilayer. This is explained as a depletion effect at the interface between tethered vesicles and the supported bilayer. Ca ions lead to transient vesicle-vesicle interactions when tethered vesicles contain negatively charged lipids, and vesicle diffusion is greatly reduced upon Ca ion addition when negatively charged lipids are present both in the supported bilayer and tethered vesicles. Both effects are interesting in their own right, and they also suggest that tethered vesicle-supported bilayer interactions are possible; this may be the origin of the reduction in D for tethered vesicles. In addition, the effects of surface defects that reversibly trap diffusing vesicles are modeled by Monte Carlo simulations. This shows that a significant reduction in D can be observed while maintaining normal diffusion behavior on the time scale of our experiments.

    View details for DOI 10.1021/la0534219

    View details for Web of Science ID 000238217000030

    View details for PubMedID 16768494

  • High yield of M-side electron transfer in mutants of Rhodobacter capsulatus reaction centers lacking the L-side bacteriopheophytin BIOCHEMISTRY Chuang, J. I., Boxer, S. G., HOLTEN, D., Kirmaier, C. 2006; 45 (12): 3845-3851

    Abstract

    We present studies on a series of photosynthetic reaction center (RC) mutants created in the background of the Rhodobacter capsulatus D(LL) mutant, in which the D helix of the M subunit has been substituted with that from the L subunit. Previous work on the D(LL) mutant in chromatophore preparations showed that RCs assembled without the bacteriopheophytin H(L) electron acceptor and performed no charge separation following light absorption. We have successfully isolated poly-His-tagged D(LL) RCs by using the detergent Deriphat 160-C and shown that the RCs are devoid of H(L). The excited state of the primary electron donor, P*, is found to have a lifetime of 180 +/- 20 ps and to decay exclusively (>95%) via internal conversion to the ground state, with no evidence for formation of any charge-separated intermediates. By additional mutation in the D(LL) background of two residues that affect the P/P+ oxidation potential and one that facilitates M-side electron transfer, we achieve an unprecedented 70% yield of P+ H(M)-, more than doubling the highest yield of this state achieved previously. This result underscores the importance of the relative free energies of P* and the charge-separated states in governing the rates and yields of electron transfer in bacterial RCs and provides a basis for systematically investigating M-side electron transfer without any competition from the native L-side pathway.

    View details for DOI 10.1021/bi0601048

    View details for Web of Science ID 000236495400001

    View details for PubMedID 16548512

  • Controlling two-dimensional tethered vesicle motion using an electric field: Interplay of electrophoresis and electro-osmosis LANGMUIR Yoshina-Ishii, C., Boxer, S. G. 2006; 22 (5): 2384-2391

    Abstract

    We recently introduced methods to tether phospholipid vesicles or proteoliposomes onto a fluid-supported lipid bilayer using DNA hybridization (Yoshina-Ishii, C.; Miller, G. P.; Kraft, M. L.; Kool, E. T.; Boxer, S. G. J. Am. Chem. Soc. 2005, 127, 1356-1357). These intact tethered vesicles diffuse in two dimensions parallel to the supporting membrane surface. In this article, we report the dynamic response of individual tethered vesicles to an electric field applied parallel to the bilayer surface. Vesicles respond to the field by moving in the direction of electro-osmotic flow, and this can be used to reversibly concentrate tethered vesicles against a barrier. By adding increasing amounts of negatively charged phosphatidylserine to the supporting bilayer to increase electro-osmosis, the electrophoretic mobility of the tethered vesicles can be increased. The electro-osmotic contribution can be modeled well by a sphere connected to a cylindrical anchor in a viscous membrane with charged headgroups. The electrophoretic force on the negatively charged tethered vesicles opposes the electro-osmotic force. By increasing the amount of negative charge on the tethered vesicle, drift in the direction of electro-osmotic flow can be slowed; at high negative charge on the tethered vesicle, motion can be forced in the direction of electrophoresis. The balance between these forces can be visualized on a patterned supporting bilayer containing negatively charged lipids that reorganize in an externally applied electric field to create a gradient of charge within a corralled region. The charge gradient at the surface creates a gradient of electro-osmotic flow, and vesicles carrying similar amounts of negative charge can be focused to a region perpendicular to the applied field where electrophoresis is balanced by electro-osmosis, away from the corral boundary. Electric fields are effective tools to direct tethered vesicles and concentrate them and to measure the tethered vesicle's electrostatic properties.

    View details for DOI 10.1021/la0526277

    View details for Web of Science ID 000235744500065

    View details for PubMedID 16489833

  • E-cadherin tethered to micropatterned supported lipid bilayers as a model for cell adhesion LANGMUIR Perez, T. D., NELSON, W. J., Boxer, S. G., Kam, L. 2005; 21 (25): 11963-11968

    Abstract

    Cell-cell adhesion is a dynamic process requiring recruitment, binding, and reorganization of signaling proteins in the plane of the plasma membrane. Here, we describe a new system for investigating how this lateral mobility influences cadherin-based cell signaling. This model is based on tethering of a GPI-modified E-cadherin protein (hEFG) to a supported lipid bilayer. In this report, membrane microfluidics and micropatterning techniques are used to adopt this tethered protein system for studies with the anchorage-dependent cells. As directly formed from proteoliposomes, hEFG exhibits a diffusion coefficient of 0.6 +/- 0.3 microm(2)/s and mobile fraction of 30-60%. Lateral structuring of the supported lipid bilayer is used to isolate mobile proteins from this mixed mobile/immobile population, and should be widely applicable to other proteins. MCF-7 cells seeded onto hEFG-containing bilayers recognize and cluster this protein, but do not exhibit cell spreading required for survival. By micropatterning small anchors into the supported lipid bilayer, we have achieved cell spreading across the bilayer surface and concurrent interaction with mobile hEFG protein. Together, these techniques will allow more detailed analysis of the cellular dynamics involved in cadherin-dependent adhesion events.

    View details for DOI 10.1021/la052264a

    View details for Web of Science ID 000233730200063

    View details for PubMedID 16316139

  • Variable incidence angle fluorescence interference contrast microscopy for z-imaging single objects BIOPHYSICAL JOURNAL Ajo-Franklin, C. M., Ganesan, P. V., Boxer, S. G. 2005; 89 (4): 2759-2769

    Abstract

    Surface-generated structured illumination microscopies interrogate the position of fluorescently labeled objects near surfaces with nanometer resolution along the z axis. However, these techniques are either experimentally cumbersome or applicable to a limited set of experimental systems. We present a new type of surface-generated structured illumination fluorescence microscopy, variable incidence angle fluorescence interference contrast microscopy (VIA-FLIC), in which the fluorescent sample is assembled above a reflective Si surface and the incidence angle of excitation light is varied by placing annular photomasks with different radii in the aperture diaphragm plane of the microscope. The variation in incidence angle alters the interference pattern of excitation light, and hence the intensity of detected fluorescence. Quantitative VIA-FLIC is tested by using a set of fluorophore-containing supported membranes separated from the Si surface by SiO2 layers of variable thicknesses. The resulting fluorescence intensity versus incidence angle curves depends on the separation from the Si surface and when fit with an appropriate model yield precise SiO2 thicknesses that are accurate with respect to the known SiO2 thicknesses. Since only a simple modification to a standard epifluorescence microscope is required, VIA-FLIC offers a versatile method to produce z-reconstructions with high resolution for a wide range of biological systems.

    View details for DOI 10.1529/biophysj.105.066738

    View details for Web of Science ID 000232147600058

    View details for PubMedID 16085775

  • Enhancement of the fluorescence of the blue fluorescent proteins by high pressure or low temperature JOURNAL OF PHYSICAL CHEMISTRY B Mauring, K., Deich, J., Rosell, F. I., McAnaney, T. B., Moerner, W. E., Boxer, S. G. 2005; 109 (26): 12976-12981

    Abstract

    Green fluorescent proteins bearing the Y66H mutation exhibit strongly blue-shifted fluorescence excitation and emission spectra. However, these blue fluorescent proteins (BFPs) have lower quantum yields of fluorescence (Phi(f) approximately 0.20), which is believed to stem from the increased conformational freedom of the smaller chromophore. We demonstrate that suppression of chromophore mobility by increasing hydrostatic pressure or by decreasing temperature can enhance the fluorescence quantum yield of these proteins without significantly affecting their absorption properties or the shape of the fluorescence spectra. Analysis of the fluorescence lifetimes in the picosecond and nanosecond regimes reveals that the enhancement of the fluorescence quantum yield is due to the inhibition of fast quenching processes. Temperature-dependent fluorescence measurements reveal two barriers ( approximately 19 and 3 kJ/mol, respectively) for the transition into nonfluorescing states. These steps are probably linked with dissociation of the hydrogen bond between the chromophore and His148 or an intervening water molecule and to the barrier for chromophore twisting in the excited state, respectively. The chromophore's hydrogen-bond equilibrium at room temperature is dominated by entropic effects, while below approximately 200 K the balance is enthalpy-driven.

    View details for DOI 10.1021/jp0448595

    View details for Web of Science ID 000230224700046

    View details for PubMedID 16852610

  • Green fluorescent protein variants as ratiometric dual emission pH sensors. 3. Temperature dependence of proton transfer BIOCHEMISTRY McAnaney, T. B., Shi, X. H., Abbyad, P., Jung, H., Remington, S. J., Boxer, S. G. 2005; 44 (24): 8701-8711

    Abstract

    In parts 1 and 2 of this series [Hanson, G. T., McAnaney, T. B., Park, E. S., Rendell, M. E. P., Yarbrough, D. K., Chu, S. Y., Xi, L. X., Boxer, S. G., Montrose, M. H., and Remington, S. J. (2002) Biochemistry 41, 15477-15488; McAnaney, T. B., Park, E. S., Hanson, G. T., Remington, S. J., and Boxer, S. G. (2002) Biochemistry 41, 15489-15494], we described the structure, excited-state dynamics, and applications of pH-sensitive, ratiometric dual emission green fluorescent protein (deGFP) variants with fluorescence emission that is modulated between blue (lambda(max) approximately equal 465 nm) and green (lambda(max) approximately equal 515 nm) depending on the pH of the bulk solvent. In this paper, we consider the energetic origin of the dual emission properties of these GFP variants by examining the temperature dependence of the steady-state absorption and fluorescence emission. In most cases, the quantum yield of the green emission decreased as the temperature was lowered, indicating that the excited-state proton transfer (ESPT) which produces the green emitting form is an activated process. The activation energies of ESPT, determined by modeling the quantum yields of both blue and green emissions between 260 and 298 K in the context of a simple photocycle, were found to be larger at low pH than at high pH. These results indicate that the ratiometric dual emission properties of deGFP mutants are due to this pH-sensitive ESPT rate, combined with a modulation of the ground-state neutral and anionic chromophore populations with pH. The time-resolved fluorescence of one of the deGFP mutants was studied in detail. The time-resolved emission spectra of this mutant are the first ultrafast spectra obtained for a GFP. These spectra demonstrate that the rising kinetics for green emission, considered a hallmark of ESPT, is the sum of the contribution from both the neutral and intermediate anionic forms of the chromophore at the probe wavelength and may not be observed in all mutants that undergo ESPT, depending on the relative contributions of the two forms.

    View details for DOI 10.1021/bi050132a

    View details for Web of Science ID 000229994900016

    View details for PubMedID 15952777

  • Probing the structure of supported membranes and tethered oligonucleotides by fluorescence interference contrast microscopy LANGMUIR Ajo-Franklin, C. M., Yoshina-Ishii, C., Boxer, S. G. 2005; 21 (11): 4976-4983

    Abstract

    Fluorescence interference contrast microscopy (FLIC) is a powerful method to structurally characterize fluorescent objects with nanometer-scale resolution in the z direction. Here we use FLIC to characterize the water layer underlying supported membranes and membrane-tethered double-stranded oligonucleotides. FLIC measurements of supported membranes containing the lipid-anchored fluorescent dye DiI in both leaflets indicate the thickness of the water layer separating the solid support and the lower lipid leaflet is 1.3 +/- 0.2 nm. Addition of cobalt(II) chloride to a DiI-supported membrane quenches the fluorescence in the top leaflet of the supported membrane; FLIC measurements of this system precisely locate the DiI to the bottom leaflet. These experiments confirm the accuracy of the model and parameters used to determine the water layer thickness, demonstrate the ability to differentiate between fluorescent objects whose average position differs by approximately 1.9 nm, and provide a widely applicable method to test the resolution of other high-z-resolution fluorescent microscopies. FLIC measurements of Alexa-labeled double-stranded oligonucleotides tethered to a supported membrane indicate that the DNA double helix is oriented perpendicular to the surface. Complications that arise from uncertainly in the orientation of the fluorophore are discussed. Several improvements in FLIC methodology are described. These stringent tests of the resolution of FLIC and the ability to unambiguously determine fluorescent lipid distribution provide structural insight on assemblies at membrane interfaces and permit the detection of even subtle changes at such interfaces.

    View details for DOI 10.1021/la0468388

    View details for Web of Science ID 000229243800034

    View details for PubMedID 15896039

  • Protonation, photobleaching, and photoactivation of yellow fluorescent protein (YFP 10C): A unifying mechanism BIOCHEMISTRY McAnaney, T. B., Zeng, W., Doe, C. F., Bhanji, N., Wakelin, S., Pearson, D. S., Abbyad, P., Shi, X. H., Boxer, S. G., Bagshaw, C. R. 2005; 44 (14): 5510-5524

    Abstract

    Yellow fluorescent protein (YFP 10C) is widely used as a probe in biology, but its complex photochemistry gives rise to unusual behavior that requires fuller definition. Here we characterize the kinetics of protonation and reversible bleaching over time scales of picoseconds to hours. Stopped-flow and pressure-jump techniques showed that protonation of the fluorescent YFP(-) anion state is two-step with a slow transition that accounts for blinking of 527 nm emission at the single molecule level on the seconds time scale. Femtosecond spectroscopy revealed that the protonated excited-state (YFPH*) decayed predominantly by a radiationless mechanism, but emission at 460 nm was detected within the first picosecond. Limited excited-state proton transfer leads to 527 nm emission characteristic of the YFP(-*) anion. Prolonged continuous wave illumination at the peak of YFP(-) absorbance (514 nm) yields, irreversibly, a weakly fluorescent product that absorbs at 390 nm. This "photobleaching" process also gives a different species (YFPHrb) that absorbs at 350/430 nm and spontaneously regenerates YFP(-) in the dark on the time scale of hours but can be photoactivated by UV light to regenerate YFP(-) within seconds, via a ground-state protonated intermediate. Using a pulsed laser for photobleaching resulted in decarboxylation of YFP as indicated by the mass spectrum. These observations are accounted for in a unifying kinetic scheme.

    View details for DOI 10.1021/bi047581f

    View details for Web of Science ID 000228252300025

    View details for PubMedID 15807545

  • Supported membrane composition analysis by secondary ion mass spectrometry with high lateral resolution BIOPHYSICAL JOURNAL Marxer, C. G., Kraft, M. L., Weber, P. K., Hutcheon, I. D., Boxer, S. G. 2005; 88 (4): 2965-2975

    Abstract

    The lateral organization of lipid components within membranes is usually investigated with fluorescence microscopy, which, though highly sensitive, introduces bulky fluorophores that might alter the behavior of the components they label. Secondary ion mass spectroscopy performed with a NanoSIMS 50 instrument also provides high lateral resolution and sensitivity, and many species can be observed in parallel without the use of bulky labels. A tightly focused beam (approximately 100 nm) of Cs ions is scanned across a sample, and up to five of the resulting small negative secondary ions can be simultaneously analyzed by a high-resolution mass spectrometer. Thin layers of (15)N- and (19)F-labeled proteins were microcontact-printed on an oxidized silicon substrate and imaged using the NanoSIMS 50, demonstrating the sensitivity and selectivity of this approach. Supported lipid bilayers were assembled on an oxidized silicon substrate, then flash-frozen and freeze-dried to preserve their lateral organization. Lipid bilayers were analyzed with the NanoSIMS 50, where the identity of each specific lipid was determined through detection of its unique secondary ions, including (12)C(1)H(-), (12)C(2)H(-), (13)C(-), (12)C(14)N(-), and (12)C(15)N(-). Steps toward obtaining quantitative composition analysis of lipid membranes that varied spatially in isotopic composition are presented. This approach has the potential to provide a composition-specific analysis of membrane organization that compliments other imaging modalities.

    View details for DOI 10.1529/biophysj.104.057257

    View details for Web of Science ID 000227986300054

    View details for PubMedID 15695628

  • General method for modification of liposomes for encoded assembly on supported bilayers JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Yoshina-Ishii, C., Miller, G. P., Kraft, M. L., Kool, E. T., Boxer, S. G. 2005; 127 (5): 1356-1357

    Abstract

    An amphiphilic oligonucleotide species ((C18)2-DNA) is presented as a generally useful reagent to display encoded tether sequences on the surface of phospholipid assemblies. (C18)2-DNA inserts into preformed vesicles and proteoliposomes of arbitrary composition, content, and origin using a simple and gentle procedure and is a significant improvement over the previously described method particularly since it allows postmodification of any phospholipid assembly without the need for special lipids carrying reactive headgroups. DNA-modified vesicles can then be tethered, via DNA hybridization, onto a supported phospholipid bilayer displaying the complementary sequence. The encoding capability of the tether can be exploited to form an array of tethered vesicles spatially defined by the DNA sequence displayed on the surface and demonstrates that (C18)2-DNA is stably associated with a membrane to allow sorting. Vesicles tethered in this way show two-dimensional mobility, reflecting the fluidity of the supporting bilayer, and promises to be a useful system with which to study vesicle-vesicle interactions.

    View details for DOI 10.1021/ja043299k

    View details for Web of Science ID 000226843900011

    View details for PubMedID 15686351

  • Vesicle adsorption and lipid bilayer formation on glass studied by atomic force microscopy LANGMUIR Schonherr, H., Johnson, J. M., Lenz, P., Frank, C. W., Boxer, S. G. 2004; 20 (26): 11600-11606

    Abstract

    The adsorption of phosphatidylcholine (PC) vesicles (30, 50, and 100 nm nominal diameters) and of dye-labeled PC vesicles (labeled with 6% Texas Red fluorophore (TR) and encapsulated carboxy fluorescein (CF)) to glass surfaces was studied by contact mode atomic force microscopy in aqueous buffer. These studies were performed in part to unravel details of the previously observed isolated rupture of dye-labeled PC vesicles on glass (Johnson, J. M.; Ha, T.; Chu, S.; Boxer, S. G. Biophys. J. 2002, 83, 3371-3379), specifically to differentiate partial rupture, that is, pore formation and leakage of entrapped dye, from full rupture to form bilayer disks. In addition, the adhesion potential of PC vesicles on glass was calculated based upon the adhesion-driven flattening of adsorbed vesicles and a newly developed theoretical model. The vesicles were found to flatten considerably upon adsorption to glass (width-to-height ratio of approximately 5), which leads to an estimate for the adhesion potential and for the critical rupture radius of 1.5 x 10(-4) J/m2 and 250 nm, respectively. Independent of vesicle size and loading with dye molecules, the adsorption of intact vesicles was observed at all concentrations below a threshold concentration, above which the formation of smooth lipid bilayers occurred. In conjunction with previous work (Johnson, J. M.; Ha, T.; Chu, S.; Boxer, S. G. Biophys. J. 2002, 83, 3371-3379), these data show that 6% TR 20 mM CF vesicles adsorb to the surface intact but undergo partial rupture in which they exchange content with the external buffer.

    View details for DOI 10.1021/la049302v

    View details for Web of Science ID 000225816800046

    View details for PubMedID 15595789

  • Patterned supported lipid bilayers and monolayers on poly(dimethylsiloxane) LANGMUIR Lenz, P., Ajo-Franklin, C. M., Boxer, S. G. 2004; 20 (25): 11092-11099

    Abstract

    A simple and practical method for patterning supported lipid bilayers on poly(dimethylsiloxane) is presented. By using electron microscopy grids to laterally control the extent of plasma oxidation, the substrate is partitioned into regions of different hydrophilicities. Addition of vesicles then results in the spontaneous formation of lipid bilayers and monolayers side-by-side on the surface, separated by regions that contain no lipid and/or a region with adhering vesicles. By using millimeter-sized plastic masks we are able to control the formation of these lipid structures on macroscopic patches by simply varying the plasma-cleaning time. For the first time, we are able to influence, in a controlled fashion, the chemical composition of a substrate in such a way that it supports fluid lipid monolayers, rejects lipid adhesion, adsorbs intact lipid vesicles, or supports fluid bilayers.

    View details for DOI 10.1021/la048450i

    View details for Web of Science ID 000225500700040

    View details for PubMedID 15568862

  • Probing excited-state electron transfer by resonance stark spectroscopy: 4 mutations near B-L in photosynthetic reaction centers perturb multiple factors that affect B-L(*)-> BL+HL- JOURNAL OF PHYSICAL CHEMISTRY B Treynor, T. P., Yoshina-Ishii, C., Boxer, S. G. 2004; 108 (35): 13523-13535

    View details for DOI 10.1021/jp048986c

    View details for Web of Science ID 000223600800060

  • Probing excited-state electron transfer by resonance stark spectroscopy: 3. Theoretical foundations and practical applications JOURNAL OF PHYSICAL CHEMISTRY B Treynor, T. P., Boxer, S. G. 2004; 108 (35): 13513-13522

    View details for DOI 10.1021/jp048988x

    View details for Web of Science ID 000223600800059

  • A theory of intervalence band stark effects JOURNAL OF PHYSICAL CHEMISTRY A Treynor, T. P., Boxer, S. G. 2004; 108 (10): 1764-1778

    View details for DOI 10.1021/jp035890u

    View details for Web of Science ID 000220094100020

  • Vibrational Stark effects calibrate the sensitivity of vibrational probes for electric fields in proteins BIOCHEMISTRY Suydam, I. T., Boxer, S. G. 2003; 42 (41): 12050-12055

    Abstract

    Infrared spectroscopy is widely used to probe local environments and dynamics in proteins. The introduction of a unique vibration at a specific site of a protein or more complex assembly offers many advantages over observing the spectra of an unmodified protein. We have previously shown that infrared frequency shifts in proteins can arise from differences in the local electric field at the probe vibration. Thus, vibrational frequencies can be used to map electric fields in proteins at many sites or to measure the change in electric field due to a perturbation. The Stark tuning rate gives the sensitivity of a vibrational frequency to an electric field, and for it to be useful, the Stark tuning rate should be as large as possible. Vibrational Stark effect spectroscopy provides a direct measurement of the Stark tuning rate and allows a quantitative interpretation of frequency shifts. We present vibrational Stark spectra of several bond types, extending our work on nitriles and carbonyls and characterizing four additional bond types (carbon-fluorine, carbon-deuterium, azide, and nitro bonds) that are potential probes for electric fields in proteins. The measured Stark tuning rates, peak positions, and extinction coefficients provide the primary information needed to design amino acid analogues or labels to act as probes of local environments in proteins.

    View details for DOI 10.1021/bi0352926

    View details for Web of Science ID 000186007000018

    View details for PubMedID 14556636

  • Arrays of mobile tethered vesicles on supported lipid bilayers JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Yoshina-Ishii, C., Boxer, S. G. 2003; 125 (13): 3696-3697

    Abstract

    We report a new system of laterally mobile, arrayed vesicles that are encoded with DNA to control tethering to fluid-supported phospholipid bilayers. The motion of individual fluorescently labeled vesicles, specifically bound, are easily visualized by fluorescence video microscopy and observed to collide reversibly on the surface. This system is an ideal model for studying interactions involving membranes, in particular integral membrane proteins.

    View details for DOI 10.1021/ja029783+

    View details for Web of Science ID 000181863200012

    View details for PubMedID 12656589

  • Polarized absorption spectra of green fluorescent protein single crystals: Transition dipole moment directions BIOCHEMISTRY Rosell, F. I., Boxer, S. G. 2003; 42 (1): 177-183

    Abstract

    Polarized absorption spectra of orthorhombic crystals of wild-type green fluorescent protein (GFP) were measured between 350 and 520 nm to obtain information on the directions of the electronic transition dipole moments ((-->)m) of the chromophore relative to the molecular axes. The transition dipole moment orientation is a basic spectroscopic parameter of relevance to biologists when interpreting Förster-type fluorescence resonance energy transfer data and for comparing absorbance and fluorescence spectra of GFP with quantum chemical calculations. Maximal extinction was obtained throughout the spectrum when the polarization direction of the electric vector of incident light was parallel to the c-axis of the crystal. The transition dipole moments were assumed to be parallel to the plane of the chromophore. With this assumption and the measured dichroic ratios in the crystals, the transition dipole moments associated with the neutral (lambda(max) = 398 nm) and anionic (lambda(max) = 478 nm) forms of the chromophore were found to subtend angles of approximately 26 degrees and 13 degrees (counterclockwise), respectively, with the vector that joins the phenolic and imidazolinone oxygen atoms of the chromophore.

    View details for DOI 10.1021/bi0266535

    View details for Web of Science ID 000180324300020

    View details for PubMedID 12515552

  • Green fluorescent protein variants as ratiometric dual emission pH sensors. 1. Structural characterization and preliminary application BIOCHEMISTRY Hanson, G. T., McAnaney, T. B., Park, E. S., Rendell, M. E., Yarbrough, D. K., Chu, S. Y., Xi, L. X., Boxer, S. G., Montrose, M. H., Remington, S. J. 2002; 41 (52): 15477-15488

    Abstract

    Novel dual emission, pH-sensitive variants of the green fluorescent protein (GFP) have been constructed and are suitable for ratiometric emission measurements in vivo. This new class of GFPs, termed deGPFs, results from substitution of wild-type residue 65 with threonine and residues 148 and/or 203 with cysteine. deGFPs display pK(a) values ranging from 6.8 to 8.0 and emission that switches from a green form (lambda(max) approximately 515 nm) to a blue form (lambda(max) approximately 460 nm) with acidifying pH. In this report we analyze in most detail the deGFP1 variant (S65T/H148G/T203C, pK(a) approximately 8.0) and the deGFP4 variant (S65T/C48S/H148C/T203C, pK(a) approximately 7.3). In the following paper [McAnaney, T. B., Park, E. S., Hanson, G. T., Remington, S. J., and Boxer, S. G. (2002) Biochemistry 41, 15489-15494], data obtained by ultrafast fluorescence upconversion spectroscopy can be described by a kinetic model that includes an excited-state proton-transfer pathway at high pH but not at low pH. Crystal structure analyses of deGFP1 at high-pH and low-pH conformations were performed to elucidate the basis for the dual emission characteristics. At low pH the structure does not contain a hydrogen bond network that would support rapid transfer of a proton from the excited state of the neutral chromophore to a suitable acceptor; hence blue emission is observed. At high pH, backbone rearrangements induced by changes in the associated hydrogen bond network permit excited-state proton transfer from the excited state of the neutral chromophore to the bulk solvent via Ser147 and bound water molecules, resulting in green emission from the anionic chromophore. Comparative analysis suggests that the basis for dual emission is elimination of the wild-type proton-transfer network by the S65T substitution, a general reduction in hydrogen-bonding opportunities, and a concomitant increase in the hydrophobic nature of the chromophore environment resulting from the cysteine substitutions. We evaluated the suitability of the deGFP4 variant for intracellular pH measurements in mammalian cells by transient expression in PS120 fibroblasts. The responses of deGFP4 and a commercially available pH-sensitive dye, SNARF-1, to changes in pH were compared in the same cells. Results show that the dynamic range of the emission ratio change is comparable between the two pH sensors over the range examined. Two-photon excitation was found to elicit a better deGFP4 fluorescent signal above cellular autofluorescence when compared to conventional confocal microscopy. Given their favorable optical characteristics, suitable pK(a)'s for the physiological pH range, and suitability for ratiometric measurements, dual emission GFPs should make excellent probes for studying pH in vivo.

    View details for DOI 10.1021/bi026609p

    View details for Web of Science ID 000180171800004

    View details for PubMedID 12501176

  • Green fluorescent protein variants as ratiometric dual emission pH sensors. 2. Excited-state dynamics BIOCHEMISTRY McAnaney, T. B., Park, E. S., Hanson, G. T., Remington, S. J., Boxer, S. G. 2002; 41 (52): 15489-15494

    Abstract

    In the preceding paper [Hanson, G. T., McAnaney, T. B., Park, E. S., Rendell, M. E. P., Yarbrough, D. K., Chu, S., Xi, L., Boxer, S. G., Montrose, M. H., and Remington, S. J. (2002) Biochemistry 41, 15477-15488], novel mutants of the green fluorescent protein (GFP) that exhibit dual steady-state emission properties were characterized structurally and discussed as potential intracellular pH probes. In this work, the excited-state dynamics of one of these new dual emission GFP variants, deGFP4 (C48S/S65T/H148C/T203C), is studied by ultrafast fluorescence upconversion spectroscopy. Following excitation of the high-energy absorption band centered at 398 nm and assigned to the neutral form of the chromophore, time-resolved emission was monitored from the excited state of both the neutral and intermediate anionic chromophores at both high and low pH and upon deuteration of exchangeable protons. The time-resolved emission dynamics and isotope effect appear to be very different from those of wild-type GFP [Chattoraj, M., King, B. A., Bublitz, G. U., and Boxer, S. G. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 8362-8367]; however, due to overlapping emission bands, the apparent difference can be analyzed quantitatively within the same framework used to describe GFP excited-state dynamics. The results indicate that the pH-sensitive steady-state emission characteristics of deGFP4 are a result of a pH-dependent modulation of the rate of excited-state proton transfer. At high pH, a rapid interconversion from the excited state of the higher energy neutral chromophore to the lower energy intermediate anionic chromophore is achieved by proton transfer. At low pH, excited-state proton transfer is slowed to the point where it is no longer rate limiting.

    View details for DOI 10.1021/bi026610o

    View details for Web of Science ID 000180171800005

    View details for PubMedID 12501177

  • Early steps of supported bilayer formation probed by single vesicle fluorescence assays BIOPHYSICAL JOURNAL Johnson, J. M., Ha, T., Chu, S., Boxer, S. G. 2002; 83 (6): 3371-3379

    Abstract

    We have developed a single vesicle assay to study the mechanisms of supported bilayer formation. Fluorescently labeled, unilamellar vesicles (30-100 nm diameter) were first adsorbed to a quartz surface at low enough surface concentrations to visualize single vesicles. Fusion and rupture events during the bilayer formation, induced by the subsequent addition of unlabeled vesicles, were detected by measuring two-color fluorescence signals simultaneously. Lipid-conjugated dyes monitored the membrane fusion while encapsulated dyes reported on the vesicle rupture. Four dominant pathways were observed, each exhibiting characteristic two-color fluorescence signatures: 1) primary fusion, in which an unlabeled vesicle fuses with a labeled vesicle on the surface, is signified by the dequenching of the lipid-conjugated dyes followed by rupture and final merging into the bilayer; 2) simultaneous fusion and rupture, in which a labeled vesicle on the surface ruptures simultaneously upon fusion with an unlabeled vesicle; 3) no dequenching, in which loss of fluorescence signal from both dyes occur simultaneously with the final merger into the bilayer; and 4) isolated rupture (pre-ruptured vesicles), in which a labeled vesicle on the surface spontaneously undergoes content loss, a process that occurs with high efficiency in the presence of a high concentration of Texas Red-labeled lipids. Vesicles that have undergone content loss appear to be more fusogenic than intact vesicles.

    View details for Web of Science ID 000180256300040

    View details for PubMedID 12496104

  • Proximal ligand motions in H93G myoglobin EUROPEAN JOURNAL OF BIOCHEMISTRY Franzen, S., Peterson, E. S., Brown, D., Friedman, J. M., Thomas, M. R., Boxer, S. G. 2002; 269 (19): 4879-4886

    Abstract

    Resonance Raman spectroscopy has been used to observe changes in the iron-ligand stretching frequency in photoproduct spectra of the proximal cavity mutant of myoglobin H93G. The measurements compare the deoxy ferrous state of the heme iron in H93G(L), where L is an exogenous imidazole ligand bound in the proximal cavity, to the photolyzed intermediate of H93G(L)*CO at 8 ns. There are significant differences in the frequencies of the iron-ligand axial out-of-plane mode nu(Fe-L) in the photoproduct spectra depending on the nature of L for a series of methyl-substituted imidazoles. Further comparison was made with the proximal cavity mutant of myoglobin in the absence of exogenous ligand (H93G) and the photoproduct of the carbonmonoxy adduct of H93G (H93G-*CO). For this case, it has been shown that H2O is the axial (fifth) ligand to the heme iron in the deoxy form of H93G. The photoproduct of H93G-*CO is consistent with a transiently bound ligand proposed to be a histidine. The data presented here further substantiate the conclusion that a conformationally driven ligand switch exists in photolyzed H93G-*CO. The results suggest that ligand conformational changes in response to dynamic motions of the globin on the nanosecond and longer time scales are a general feature of the H93G proximal cavity mutant.

    View details for DOI 10.1046/j.1432-1033.2002.03193.x

    View details for Web of Science ID 000178275200018

    View details for PubMedID 12354119

  • Probing protein electrostatics with a synthetic fluorescent amino acid SCIENCE Cohen, B. E., McAnaney, T. B., Park, E. S., Jan, Y. N., Boxer, S. G., Jan, L. Y. 2002; 296 (5573): 1700-1703

    Abstract

    Electrostatics affect virtually all aspects of protein structure and activity and are particularly important in proteins whose primary function is to stabilize charge. Here we introduce a fluorescent amino acid, Aladan, which can probe the electrostatic character of a protein at multiple sites. Aladan is exceptionally sensitive to the polarity of its surroundings and can be incorporated site-selectively at buried and exposed sites, in both soluble and membrane proteins. Steady-state and time-resolved fluorescence measurements of Aladan residues at different buried and exposed sites in the B1 domain of protein G suggest that its interior is polar and heterogeneous.

    View details for Web of Science ID 000175976200063

    View details for PubMedID 12040199

  • Micropattern formation in supported lipid membranes ACCOUNTS OF CHEMICAL RESEARCH Groves, J. T., Boxer, S. G. 2002; 35 (3): 149-157

    Abstract

    Phospholipid vesicles exhibit a natural tendency to fuse and assemble into a continuous single bilayer membrane on silica and several other substrate materials. The resulting supported membrane maintains many of the physical and biological characteristics of free membranes, including lateral fluidity. Recent advances, building on the supported membrane configuration, have created a wealth of opportunities for the manipulation, control, and analysis of membranes and the reaction environments they provide. The work reviewed in this Account, which can be broadly characterized as the science and technology of membrane patterning, contains three basic components: lateral diffusion control (barriers), membrane deposition techniques (microarrays), and electric field-induced lateral reorganization. Collectively, these preparative and analytical patterned membrane techniques offer a broad experimental platform for the study and utilization of lipid membranes.

    View details for DOI 10.1021/ar950039m

    View details for Web of Science ID 000174597800002

    View details for PubMedID 11900518

  • FTIR and resonance Raman studies of nitric oxide binding to H93G cavity mutants of myoglobin BIOCHEMISTRY Thomas, M. R., Brown, D., Franzen, S., Boxer, S. G. 2001; 40 (49): 15047-15056

    Abstract

    Nitric oxide (NO) binds to the myoglobin (Mb) cavity mutant, H93G, forming either a five- or six-coordinate Fe-NO complex. The H93G mutation eliminates the covalent attachment between the protein and the proximal ligand, allowing NO to bind H93G possibly from the proximal side of the heme rather than the typical diatomic binding pocket on the distal side. The question of whether NO binds on the distal or proximal side was addressed by FTIR spectroscopy of the N-O vibrational frequency nuN(-O) for a set of Mb mutants that perturb the electrostatic environment of the heme pocket. Vibrational spectra of five- and six-coordinate MbNO complexes indicate that nu(N-O) shifts (by as much as 26 cm(-1)) to higher energies for the distal mutants H64V and H64V/H93G relative to the energies of wild-type and H93G MbNO, while nu(N-O) is not affected by the proximal side mutation S92A/H93G. This result suggests that NO binds on the distal side of heme in the five- and six-coordinate MbNO complexes of H93G. Additionally, values of the Fe-NO vibrational frequency nu(Fe-NO) as measured by resonance Raman spectroscopy are reported for the distal and proximal double mutants of H93G. These results suggest that nu(Fe-NO) is not very sensitive to mutations that perturb the electrostatic environment of the heme pocket, leading to the observation that nu(N-O) and nu(Fe-NO) are not quantitatively correlated for the MbNO complexes presented here. Furthermore, nu(N-O) and nu(Fe-NO) do not correlate well with equilibrium constants for imidazole binding to the five-coordinate MbNO complexes of the H93G double mutants. The data presented here do not appear to support the presence of pi-back-bonding or an inverse trans effect of NO binding in Mb mutants that alter the electrostatic environment of the heme pocket.

    View details for Web of Science ID 000172608100037

    View details for PubMedID 11732927

  • Electrostatic and conformational effects on the electronic structures of distortional isomers of a mixed-valence binuclear Cu complex INORGANIC CHEMISTRY Franzen, S., Miskowski, V. M., Shreve, A. P., Wallace-Williams, S. E., Woodruff, W. H., Ondrias, M. R., Barr, M. E., Moore, L., Boxer, S. G. 2001; 40 (25): 6375-6382

    Abstract

    The electronic structure of the binuclear copper complex [Cu(2)(L)](3+) [L = N(CH(2)CH(2)N(H)CH(2)CH(2)N(H)CH(2)CH(2))(3)N] has been investigated by resonance Raman and electroabsorption spectroscopy. Crystallographic Cu(2) distances of 2.364(1) and 2.415(1) A determined for the nitrate and acetate salts, respectively, are consistent with a substantial metal-metal interaction. The Cu-Cu bonding interaction in the binuclear complex is modulated both in the solid state and in solution by the ligand environment through coupling to ligand torsional modes that are, in turn, stabilized by hydrogen bonding. Electroabsorption data on the three major visible and near-infrared electronic transitions of Cu(2)L, lambda(max) (epsilon(max)) = 1000 nm ( approximately 1200 M(-1) cm(-1)), 748 nm (5600 M(-1) cm(-1)), and 622 nm (3350 M(-1) cm(-1)), reveal a difference dipole moment between the ground and excited states (Deltamu(A)) because of symmetry breaking. The difference polarizability for all three of the transitions is negative, indicating that the ground state is more polarizable than the excited state. A general model to explain this behavior in terms of the proximity of accessible transitions involving copper d electrons is proposed to explain the larger polarizability of the ground state. Raman excitation profiles (REPs) provide evidence for multiple conformational states of [Cu(2)(L)](3+). Separate REPs were obtained for each of the components of the two major Raman bands for nu(1) (a Cu-Cu stretching mode) and nu(2) (a Cu-Cu-N(eq) bending mode). The Raman data along with quantum chemical ZINDO/S CI calculations provide evidence for isomeric forms of Cu(2)L with strong coupling between the conformation of L and the Cu-Cu bond length.

    View details for DOI 10.1021/ic010494g

    View details for Web of Science ID 000172495000006

    View details for PubMedID 11720490

  • High refractive index substrates for fluorescence microscopy of biological interfaces with high z contrast PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ajo-Franklin, C. M., Kam, L., Boxer, S. G. 2001; 98 (24): 13643-13648

    Abstract

    Total internal reflection fluorescence microscopy is widely used to confine the excitation of a complex fluorescent sample very close to the material on which it is supported. By working with high refractive index solid supports, it is possible to confine even further the evanescent field, and by varying the angle of incidence, to obtain quantitative information on the distance of the fluorescent object from the surface. We report the fabrication of hybrid surfaces consisting of nm layers of SiO(2) on lithium niobate (LiNbO(3), n = 2.3). Supported lipid bilayer membranes can be assembled and patterned on these hybrid surfaces as on conventional glass. By varying the angle of incidence of the excitation light, we are able to obtain fluorescent contrast between 40-nm fluorescent beads tethered to a supported bilayer and fluorescently labeled protein printed on the surface, which differ in vertical position by only tens of nm. Preliminary experiments that test theoretical models for the fluorescence-collection factor near a high refractive index surface are presented, and this factor is incorporated into a semiquantitative model used to predict the contrast of the 40-nm bead/protein system. These results demonstrate that it should be possible to profile the vertical location of fluorophores on the nm distance scale in real time, opening the possibility of many experiments at the interface between supported membranes and living cells. Improvements in materials and optical techniques are outlined.

    View details for Web of Science ID 000172328100033

    View details for PubMedID 11717428

  • Spatially localized generation of nucleotide sequence-specific DNA damage PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Oh, D. H., King, B. A., Boxer, S. G., Hanawalt, P. C. 2001; 98 (20): 11271-11276

    Abstract

    Psoralens linked to triplex-forming oligonucleotides (psoTFOs) have been used in conjunction with laser-induced two-photon excitation (TPE) to damage a specific DNA target sequence. To demonstrate that TPE can initiate photochemistry resulting in psoralen-DNA photoadducts, target DNA sequences were incubated with psoTFOs to form triple-helical complexes and then irradiated in liquid solution with pulsed 765-nm laser light, which is half the quantum energy required for conventional one-photon excitation, as used in psoralen + UV A radiation (320-400 nm) therapy. Target DNA acquired strand-specific psoralen monoadducts in a light dose-dependent fashion. To localize DNA damage in a model tissue-like medium, a DNA-psoTFO mixture was prepared in a polyacrylamide gel and then irradiated with a converging laser beam targeting the rear of the gel. The highest number of photoadducts formed at the rear while relatively sparing DNA at the front of the gel, demonstrating spatial localization of sequence-specific DNA damage by TPE. To assess whether TPE treatment could be extended to cells without significant toxicity, cultured monolayers of normal human dermal fibroblasts were incubated with tritium-labeled psoralen without TFO to maximize detectable damage and irradiated by TPE. DNA from irradiated cells treated with psoralen exhibited a 4- to 7-fold increase in tritium activity relative to untreated controls. Functional survival assays indicated that the psoralen-TPE treatment was not toxic to cells. These results demonstrate that DNA damage can be simultaneously manipulated at the nucleotide level and in three dimensions. This approach for targeting photochemical DNA damage may have photochemotherapeutic applications in skin and other optically accessible tissues.

    View details for Web of Science ID 000171237100054

    View details for PubMedID 11572980

  • F-19 NMR of trifluoroacectyl-labeled cysteine mutants of myoglobin: Structural probes of nitric oxide bound to the H93G cavity mutant BIOCHEMISTRY Thomas, M. R., Boxer, S. G. 2001; 40 (29): 8588-8596

    Abstract

    Nitric oxide (NO) binds to the myoglobin (Mb) cavity mutant, H93G, forming either a 5- or 6-coordinate Fe--NO heme complex. The H93G mutation replaces the proximal histidine of Mb with glycine, allowing exogenous ligands to occupy the proximal binding site. In the absence of the covalently attached proximal ligand, NO could bind to H93G from the proximal side of the heme rather than the typical diatomic binding pocket on the distal side when the 5-coordinate complex forms. The question of whether NO binds on the distal or proximal side was addressed by (19)F NMR. Site-directed mutagenesis was used to introduce unique cysteine residues at the protein surface on either the distal (S58C) or proximal (L149C) side, approximately equidistant from and perpendicular to the heme plane of both wild-type and H93G Mb. The cysteine thiols were alkylated with 3-bromo-1,1,1-trifluoroacetone to attach a trifluoroacetyl group at the mutation site. (19)F NMR spectra of 5-coordinate, NO bound S58C/H93G and L149C/H93G double mutants depict peaks with line widths of 100 and 23 Hz, respectively. As fluorine peaks broaden with increasing proximity to paramagnetic centers, such as 5-coordinate Fe--NO, the (19)F NMR data are consistent with NO binding in the distal heme pocket of H93G, even in the absence of a sixth axial ligand. Additionally, (19)F NMR spectra are reported for deoxy, oxy, CO, met CN, and met H(2)O forms of the labeled cysteine mutants. These results demonstrate that the fluorine probes are sensitive to subtle conformational changes in the protein structure due to ligation and oxidation state changes of the heme iron in Mb.

    View details for Web of Science ID 000170057200021

    View details for PubMedID 11456499

  • Cell adhesion to protein-micropatterned-supported lipid bilayer membranes JOURNAL OF BIOMEDICAL MATERIALS RESEARCH Kam, L., Boxer, S. G. 2001; 55 (4): 487-495

    Abstract

    A new method for constructing controlled interfaces between cells and synthetic supported lipid bilayer membranes is reported. Microcontact printing is used to define squares and grid lines of fibronectin onto glass, which subsequently direct the self-assembly of fluid lipid bilayers onto the complementary, uncoated regions of the surface. Features of fibronectin as small as 5 microm effectively control the lateral organization of the lipid bilayers. These fibronectin barriers also facilitate the adhesion of endothelial cells, which exhibit minimal adhesion to fluid supported lipid bilayers alone. Cells selectively adhere to the features of fibronectin, spanning over and exposing the cells to the intervening regions of supported lipid bilayer. Cell spreading is correlated with both the geometry and dimensions of the fibronectin barriers. Importantly, lipids underlying adherent cells are laterally mobile, suggesting that, in contrast to the regions of fibronectin, cells were not in direct contact with the supported membrane. Protein micropatterning thus provides a valuable tool for controlling supported membranes and for juxtaposing anchorage-dependent cells with lipid bilayers. These systems should be generally useful for studying specific interactions between cells and biomolecules incorporated into supported membranes, and as an approach for integrating living cells with synthetic, laterally complex surfaces.

    View details for Web of Science ID 000168199600006

    View details for PubMedID 11288076

  • Photophysics of DsRed, a red fluorescent protein, from the ensemble to the single-molecule level JOURNAL OF PHYSICAL CHEMISTRY B Lounis, B., Deich, J., Rosell, F. I., Boxer, S. G., Moerner, W. E. 2001; 105 (21): 5048-5054
  • A photolysis-triggered heme ligand switch in H93G myoglobin BIOCHEMISTRY Franzen, S., Bailey, J., Dyer, R. B., Woodruff, W. H., Hu, R. B., Thomas, M. R., Boxer, S. G. 2001; 40 (17): 5299-5305

    Abstract

    Resonance Raman spectroscopy and step-scan Fourier transform infrared (FTIR) spectroscopy have been used to identify the ligation state of ferrous heme iron for the H93G proximal cavity mutant of myoglobin in the absence of exogenous ligand on the proximal side. Preparation of the H93G mutant of myoglobin has been previously reported for a variety of axial ligands to the heme iron (e.g., substituted pyridines and imidazoles) [DePillis, G., Decatur, S. M., Barrick, D., and Boxer, S. G. (1994) J. Am. Chem. Soc. 116, 6981-6982]. The present study examines the ligation states of heme in preparations of the H93G myoglobin with no exogenous ligand. In the deoxy form of H93G, resonance Raman spectroscopic evidence shows water to be the axial (fifth) ligand to the deoxy heme iron. Analysis of the infrared C-O and Raman Fe-C stretching frequencies for the CO adduct indicates that it is six-coordinate with a histidine trans ligand. Following photolysis of CO, a time-dependent change in ligation is evident in both step-scan FTIR and saturation resonance Raman spectra, leading to the conclusion that a conformationally driven ligand switch exists in the H93G protein. In the absence of exogenous nitrogenous ligands, the CO trans effect stabilizes endogenous histidine ligation, while conformational strain favors the dissociation of histidine following photolysis of CO. The replacement of histidine by water in the five-coordinate complex is estimated to occur in < 5 micros. The results demonstrate that the H93G myoglobin cavity mutant has potential utility as a model system for studying the conformational energetics of ligand switching in heme proteins such as those observed in nitrite reductase, guanylyl cyclase, and possibly cytochrome c oxidase.

    View details for DOI 10.1021/bi0023403

    View details for Web of Science ID 000168435100023

    View details for PubMedID 11318654

  • Polymer-supported lipid bilayers on benzophenone-modified substrates BIOMACROMOLECULES Shen, W. W., Boxer, S. G., Knoll, W., Frank, C. W. 2001; 2 (1): 70-79

    Abstract

    Solid-supported lipid membranes are important for their roles in fundamental biophysical research as well as in applications such as biosensors. In our study, lipopolymers containing alkyl side chains were synthesized and a mixture of the lipopolymer and free lipids was preorganized at the air-water interface and then transferred to a solid substrate using the Langmuir-Blodgett technique. A photochemical reaction between a substrate-functionalized benzophenone and C-H bonds on the lipopolymer was used to attach the lipopolymers to the substrate. The final assembly of the membrane was completed by vesicle fusion. Langmuir film experiments at the air-water interface indicate tighter molecular packing for the lipopolymers with 28 mol % alkyl side chains than for the ones with 22 mol %. Atomic force microscopy images point to phase separation of lipopolymers on the substrates due to their dewetting from hydrophobic surfaces. However, a mixture of lipopolymers and free lipids formed a smooth film on the same substrate. After the addition of the second lipid layer on the lipopolymer/free lipid layer, the fluorescence images of the polymer-supported bilayer suggested that the distal lipid layer is homogeneous on the micrometer scale. The relaxation of the fluorescent probe lipids was analyzed after application of an electric field to determine their diffusion coefficient; the distal lipid layer was mobile with an average diffusion coefficient of approximately 0.1 microm(2)/s. Moreover, the immobile fraction of the lipids in the distal layer was estimated to be around 15%.

    View details for DOI 10.1021/bm005581z

    View details for Web of Science ID 000168755800011

    View details for PubMedID 11749157

  • The H93G myoglobin cavity mutant as a versatile template for modeling heme proteins: Ferrous, ferric, and ferryl mixed-ligand complexes with imidazole in the cavity INORGANIC CHEMISTRY Pond, A. E., Roach, M. P., Thomas, M. R., Boxer, S. G., Dawson, J. H. 2000; 39 (26): 6061-6066

    Abstract

    One of the difficulties in preparing accurate ambient-temperature model complexes for heme proteins, particularly in the ferric state, has been the generation of mixed-ligand adducts: complexes with different ligands on either side of the heme. The difference in the accessibility of the two sides of the heme in the H93G cavity mutant of myoglobin (Mb) provides a potential general solution to this problem. To demonstrate the versatility of H93G Mb for the preparation of heme protein models, numerous mixed-ligand adducts of ferrous, ferric, and ferryl imidazole-ligated H93G (H93G(Im) Mb) have been prepared. The complexes have been characterized by electronic absorption and magnetic circular dichroism (MCD) spectroscopy in comparison to analogous derivatives of wild type Mb. The starting ferric H93G(Im) Mb state spectroscopically resembles wild-type ferric Mb as expected for a complex containing a single imidazole in the proximal cavity and water bound on the distal side. Addition of a sixth ligand to ferric H93G(Im) Mb, whether charge neutral (imidazole) or anionic (cyanide and azide), results in formation of six-coordinate low-spin complexes with MCD characteristics similar to those of parallel derivatives of wild-type ferric Mb. Reduction of ferric H93G(Im) Mb and subsequent exposure to either CO, NO, or O2 produces ferrous complexes (deoxy, CO, NO, and O2) that consistently exhibit MCD spectra similar to the analogous ferrous species of wild-type ferrous Mb. Most interestingly, reaction of ferric H93G(Im) Mb with H2O2 results in the formation of a stable high-valent oxoferryl complex with MCD characteristics that are essentially identical to those of oxoferryl wild-type Mb. The generation of such a wide array of mixed-ligand heme complexes demonstrates the efficacy of the H93G Mb cavity mutant as a template for the preparation of heme protein model complexes.

    View details for Web of Science ID 000166019200023

    View details for PubMedID 11151505

  • Molecular transport and organization in supported lipid membranes CURRENT OPINION IN CHEMICAL BIOLOGY Boxer, S. G. 2000; 4 (6): 704-709

    Abstract

    The mechanism by which vesicles spontaneously form supported lipid bilayer membranes on glass surfaces is becoming better understood and this knowledge is the basis of a technology of patterning membrane arrays and controlling composition. Controlled interactions between supported membranes and cells, particularly from the immune system, provide direct insight into cell-cell surface interactions.

    View details for Web of Science ID 000165854000016

    View details for PubMedID 11102877

  • Excited-state electronic asymmetry of the special pair in photosynthetic reaction center mutants: Absorption and stark spectroscopy BIOCHEMISTRY Moore, L. J., Zhou, H. L., Boxer, S. G. 1999; 38 (37): 11949-11960

    Abstract

    The electronic absorption line shape and Stark spectrum of the lowest energy Q(y)() transition of the special pair in bacterial reaction centers contain a wealth of information on mixing with charge transfer states and electronic asymmetry. Both vary greatly in mutants that perturb the chemical composition of the special pair, such as the heterodimer mutants, and in mutants that alter interactions between the special pair and the surrounding reaction center protein, such as those that add or remove hydrogen bonds. The conventional and higher-order Stark spectra of a series of mutants are presented with the aim of developing a systematic description of the electronic structure of the excited state of the special pair that initiates photosynthetic charge separation. The mutants L168HF, M197FH, L131LH and L131LH/M160LH/M197FH are known to have different hydrogen-bonding patterns to the special pair; however, they exhibit Stark effects that are very similar to wild type. By contrast, the addition of a hydrogen bond to the M-side keto carbonyl group of the special pair in M160LH greatly affects both the absorption and Stark spectra. The heterodimer special pairs, L173HL and M202HL, exhibit much larger Stark effects than wild type, with the greatest effect in the M-side mutant. Double mutants that combine the M-side heterodimer and a hydrogen-bond addition to the L-side of the special pair decrease the magnitude of the Stark effect. These results suggest that the electronic asymmetry of the dimer can be perturbed either by the formation of a heterodimer or by adding or deleting a hydrogen bond to a keto carbonyl group. From the pattern observed, it is concluded that the charge transfer state P(L)(+)P(M)(-) has a larger influence on the excited state of the dimer in wild type than the P(L)(-)P(M)(+)charge transfer state. Furthermore, asymmetry can be varied continuously, from extreme cases in which the heterodimer and hydrogen-bond effects work together, to cases in which hydrogen bonding offsets the effects of the heterodimer, to cases in which the homodimer is perturbed by hydrogen bonds. This leads to a unified model for understanding the effects of perturbations on the electronic symmetry of the special pair, and this can be connected with perturbations on the properties of many other systems such as donor-acceptor-substituted polyenes.

    View details for Web of Science ID 000082834800011

    View details for PubMedID 10508398

  • Hydrogen bonding modulates binding of exogenous ligands in a myoglobin proximal cavity mutant BIOCHEMISTRY Decatur, S. M., Belcher, K. L., Rickert, P. K., Franzen, S., Boxer, S. G. 1999; 38 (34): 11086-11092

    Abstract

    In the sperm whale myoglobin mutant H93G, the proximal histidine is replaced by glycine, leaving a cavity in which exogenous imidazole can bind and ligate the heme iron (Barrick, D. (1994) Biochemistry 33, 6545-6554). Structural studies of this mutant suggest that serine 92 may play an important role in imidazole binding by serving as a hydrogen bond acceptor. Serine 92 is highly conserved in myoglobins, forming a well-characterized weak hydrogen bond with the proximal histidine in the native protein. We have probed the importance of this hydrogen bond through studies of the double mutants S92A/H93G and S92T/H93G incorporating exogenous imidazole and methylimidazoles. (1)H NMR spectra reveal that loss of the hydrogen bond in S92A/H93G does not affect the conformation of the bound imidazole. However, the binding constants for imidazoles to the ferrous nitrosyl complex of S92A/H93G are much weaker than in H93G. These results are discussed in terms of hydrogen bonding and steric packing within the proximal cavity. The results also highlight the importance of the trans diatomic ligand in altering the binding and sensitivity to perturbation of the ligand in the proximal cavity.

    View details for Web of Science ID 000082342600020

    View details for PubMedID 10460164

  • Brownian ratchets: Molecular separations in lipid bilayers supported on patterned arrays SCIENCE van Oudenaarden, A., Boxer, S. G. 1999; 285 (5430): 1046-1048

    Abstract

    Brownian ratchets use a time-varying asymmetric potential that can be applied to separate diffusing particles or molecules. A new type of Brownian ratchet, a geometrical Brownian ratchet, has been realized. Charged, fluorescently labeled phospholipids in a two-dimensional fluid bilayer were driven in one direction by an electric field through a two-dimensional periodic array of asymmetric barriers to lateral diffusion fabricated from titanium oxide on silica. Diffusion spreads the phospholipid molecules in the orthogonal direction, and the asymmetric barriers rectify the Brownian motion, causing a directional transport of molecules. The geometrical ratchet can be used as a continuous molecular sieve to separate mixtures of membrane-associated molecules that differ in electrophoretic mobility and diffusion coefficient.

    View details for Web of Science ID 000082033100039

    View details for PubMedID 10446046

  • Assignment of the heme axial ligand(s) for the ferric myoglobin (H93G) and heme oxygenase (H25A) cavity mutants as oxygen donors using magnetic circular dichroism BIOCHEMISTRY Pond, A. E., Roach, M. P., Sono, M., Rux, A. H., Franzen, S., Hu, R., Thomas, M. R., Wilks, A., Dou, Y., Ikeda-Saito, M., de Montelano, P. R., Woodruff, W. H., Boxer, S. G., Dawson, J. H. 1999; 38 (23): 7601-7608

    Abstract

    UV-visible absorption and magnetic circular dichroism (MCD) data are reported for the cavity mutants of sperm whale H93G myoglobin and human H25A heme oxygenase in their ferric states at 4 degreesC. Detailed spectral analyses of H93G myoglobin reveal that its heme coordination structure has a single water ligand at pH 5.0, a single hydroxide ligand at pH 10.0, and a mixture of species at pH 7.0 including five-coordinate hydroxide-bound, and six-coordinate structures. The five-coordinate aquo structure at pH 5 is supported by spectral similarity to acidic horseradish peroxidase (pH 3.1), whose MCD data are reported herein for the first time, and acidic myoglobin (pH 3.4), whose structures have been previously assigned by resonance Raman spectroscopy. The five-coordinate hydroxide structure at pH 10.0 is supported by MCD and resonance Raman data obtained here and by comparison with those of other known five-coordinate oxygen donor complexes. In particular, the MCD spectrum of alkaline ferric H93G myoglobin is strikingly similar to that of ferric tyrosinate-ligated human H93Y myoglobin, whose MCD data are reported herein for the first time, and that of the methoxide adduct of ferric protoporphyrin IX dimethyl ester (FeIIIPPIXDME). Analysis of the spectral data for ferric H25A heme oxygenase at neutral pH in the context of the spectra of other five-coordinate ferric heme complexes with proximal oxygen donor ligands, in particular the p-nitrophenolate and acetate adducts of FeIIIPPIXDME, is most consistent with ligation by a carboxylate group of a nearby glutamyl (or aspartic) acid residue.

    View details for Web of Science ID 000080911600022

    View details for PubMedID 10360958

  • Electric field-induced critical demixing in lipid bilayer membranes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Groves, J. T., Boxer, S. G., McConnell, H. M. 1998; 95 (3): 935-938

    Abstract

    Electric fields can induce lateral reorganization of lipids in fluid bilayer membranes. The resulting concentration profiles readily are observed in planar-supported bilayers by epifluorescence microscopy. When a fluorescently labeled lipid was used to probe the field-induced separation of cardiolipin from egg-phosphatidylcholine, an enhanced sensitivity to the electric field was observed that is attributed to a critical demixing effect. A thermodynamic model of the system was used to analyze the results. The observed concentration profiles can be understood if the lipid mixture has a critical temperature equal to 75 degrees K. The steady-state distribution of lipids under the influence of an electric field is very sensitive to demixing effects, even at temperatures well above the critical temperature for spontaneous phase separation, and this may have significant consequences for organization and structural changes in natural cell membranes.

    View details for Web of Science ID 000071878500026

    View details for PubMedID 9448263

  • Electric field-induced reorganization of two-component supported bilayer membranes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Groves, J. T., Boxer, S. G., MCCONNEL, H. M. 1997; 94 (25): 13390-13395

    Abstract

    Application of electric fields tangent to the plane of a confined patch of fluid bilayer membrane can create lateral concentration gradients of the lipids. A thermodynamic model of this steady-state behavior is developed for binary systems and tested with experiments in supported lipid bilayers. The model uses Flory's approximation for the entropy of mixing and allows for effects arising when the components have different molecular areas. In the special case of equal area molecules the concentration gradient reduces to a Fermi-Dirac distribution. The theory is extended to include effects from charged molecules in the membrane. Calculations show that surface charge on the supporting substrate substantially screens electrostatic interactions within the membrane. It also is shown that concentration profiles can be affected by other intermolecular interactions such as clustering. Qualitative agreement with this prediction is provided by comparing phosphatidylserine- and cardiolipin-containing membranes.

    View details for Web of Science ID A1997YK82500007

    View details for PubMedID 9391034

  • Crystal structure and photodynamic behavior of the blue emission variant Y66H/Y145F of green fluorescent protein BIOCHEMISTRY Wachter, R. M., King, B. A., Heim, R., Kallio, K., Tsien, R. Y., Boxer, S. G., Remington, S. J. 1997; 36 (32): 9759-9765

    Abstract

    The crystal structure of a blue emission variant (Y66H/Y145F) of the Aequorea victoria green fluorescent protein has been determined by molecular replacement and the model refined. The crystallographic R-factor is 18.1% for all data from 20 to 2.1 A, and the model geometry is excellent. The chromophore is non-native and is autocatalytically generated from the internal tripeptide Ser65-His66-Gly67. The final electron density maps indicate that the formation of the chromophore is complete, including 1,2 dehydration of His66 as indicated by the planarity of the chromophore. The chromophore is in the cis conformation, with no evidence for any substantial fraction of the trans configuration or uncyclized apoprotein, and is well-shielded from bulk solvent by the folded protein. These characteristics indicate that the machinery for production of the chromophore from a buried tripeptide unit is not only intact but also highly efficient in spite of a major change in chromophore chemical structure. Nevertheless, there are significant rearrangements in the hydrogen bond configuration around the chromophore as compared to wild-type, indicating flexibility of the active site. pH titration of the intact protein and the chromopeptide (pKa1 = 4.9 +/- 0.1, pKa2 = 12.0 +/- 0.1) suggests that the predominant form of the chromophore in the intact protein is electrically neutral. In contrast to the wild-type protein [Chattoraj, M., King, B. A., Bublitz, G. U., & Boxer, S. G. (1996) Proc. Natl. Acad. Sci. U.S.A., 8362-8367], femtosecond fluorescence up-conversion spectroscopy of the intact protein and a partially deuterated form strongly suggests that excited-state proton transfer is not coupled to fluorescence emission.

    View details for Web of Science ID A1997XQ76000014

    View details for PubMedID 9245407

  • Temperature dependence of the Q(y) resonance Raman spectra of bacteriochlorophylls, the primary electron donor, and bacteriopheophytins in the bacterial photosynthetic reaction center BIOCHEMISTRY Cherepy, N. J., Shreve, A. P., Moore, L. J., Boxer, S. G., Mathies, R. A. 1997; 36 (28): 8559-8566

    Abstract

    Qy-excited resonance Raman spectra of the accessory bacteriochlorophylls (B), the bacteriopheophytins (H), and the primary electron donor (P) in the bacterial photosynthetic reaction center (RC) of Rhodobacter sphaeroides have been obtained at 95 and 278 K. Frequency and intensity differences are observed in the low-frequency region of the P vibrational spectrum when the sample is cooled from 278 to 95 K. The B and H spectra exhibit minimal changes of frequencies and relative intensities as a function of temperature. The mode patterns in the Raman spectra of B and H differ very little from Raman spectra of the chromophores in vitro. The Raman scattering cross sections of B and H are 6-7 times larger than those for analogous modes of P at 278 K. The cross sections of B and of H are 3-4 times larger at 95 K than at 278 K, while the cross sections of P are approximately constant with temperature. The temperature dependence of the Raman cross sections for B and H suggests that pure dephasing arising from coupling to low-frequency solvent/protein modes is important in the damping of their excited states. The weak Raman cross sections of the special pair suggest that the excited state of P is damped by very rapid (<30 fs) electronic relaxation processes. These resonance Raman spectra provide information for developing multimode vibronic models of the excited-state structure and dynamics of the chromophores in the RC.

    View details for Web of Science ID A1997XK85600016

    View details for PubMedID 9214301

  • Large molecular third-order optical nonlinearities in polarized carotenoids SCIENCE Marder, S. R., Torruellas, W. E., BLANCHARDDESCE, M., Ricci, V., Stegeman, G. I., GILMOUR, S., Bredas, J. L., Li, J., Bublitz, G. U., Boxer, S. G. 1997; 276 (5316): 1233-1236

    Abstract

    Garito and co-workers have suggested a mechanism to dramatically increase the second hyperpolarizability, gamma, in linear pi-electron-conjugated molecules. Polarization is introduced that leads to a difference between the dipole moments of the molecule's ground state and excited state. Here a series of carotenoids was examined that had increasing intramolecular charge transfer (ICT) from the polyenic chain to the acceptor moiety in the ground state, and gamma was measured for these compounds as a function of wavelength by third-harmonic generation. The compound with the greatest ICT exhibited a 35-fold enhancement of gammamax (the gamma measured at the peak of the three-photon resonance) relative to the symmetric molecule beta-carotene, which itself has one of the largest third-order nonlinearities known. Stark spectroscopic measurements revealed the existence of a large difference dipole moment, Delta mu, between the ground and excited state. Quantum-chemical calculations underline the importance of interactions involving states with large Delta mu.

    View details for Web of Science ID A1997XA49700035

    View details for PubMedID 9157876

  • Electronic and nuclear dynamics of the accessory bacteriochlorophylls in bacterial photosynthetic reaction centers from resonance Raman intensities JOURNAL OF PHYSICAL CHEMISTRY B Cherepy, N. J., Shreve, A. P., Moore, L. J., Boxer, S. G., Mathies, R. A. 1997; 101 (16): 3250-3260
  • On the origin of heme absorption band shifts and associated protein structural relaxation in myoglobin following flash photolysis JOURNAL OF BIOLOGICAL CHEMISTRY Franzen, S., Boxer, S. G. 1997; 272 (15): 9655-9660

    Abstract

    The role of the protein structural change monitored by absorption band shifts following flash photolysis of CO from myoglobin is discussed in terms of structure-function relationships. Evidence is presented that the Soret band shift does not depend primarily on the covalent linkage of the heme iron to the protein by using the mutation H93G(L) in which the proximal histidine 93 is replaced by glycine and an exogenous ligand L, which coordinates the heme iron but is not covalently bound to the globin. While CO rebinding kinetics depend strongly on the nature of the exogenous ligand L in H93G(L), the magnitude and time evolution of the Soret band shift in a viscous buffer on the nanosecond time scale are hardly perturbed in all cases studied. Comparison of the Soret band and charge transfer Band III shifts demonstrates that both have a similar time dependence on the nanosecond to microsecond time scale following flash photolysis in viscous solvents. We conclude that the nonexponential kinetics of protein relaxation probed by transient absorption band position shifts involves primarily distal coordinates prior to ligand escape. This result agrees with earlier measurements of Soret band shifts in distal pocket mutants of myoglobin (1). We suggest that the band shifts are primarily a response to changes in the electrostatic field around the heme (a transient Stark shift) associated with changes in protein structure that occur following ligand photodissociation.

    View details for Web of Science ID A1997WU03900013

    View details for PubMedID 9092494

  • Micropatterning fluid lipid bilayers on solid supports SCIENCE Groves, J. T., Ulman, N., Boxer, S. G. 1997; 275 (5300): 651-653

    Abstract

    Lithographically patterned grids of photoresist, aluminum oxide, or gold on oxidized silicon substrates were used to partition supported lipid bilayers into micrometer-scale arrays of isolated fluid membrane corrals. Fluorescently labeled lipids were observed to diffuse freely within each membrane corral but were confined by the micropatterned barriers. The concentrations of fluorescent probe molecules in individual corrals were altered by selective photobleaching to create arrays of fluid membrane patches with differing compositions. Application of an electric field parallel to the surface induced steady-state concentration gradients of charged membrane components in the corrals. In addition to producing patches of membrane with continuously varying composition, these gradients provide an intrinsically parallel means of acquiring information about molecular properties such as the diffusion coefficient in individual corrals.

    View details for Web of Science ID A1997WF07700035

    View details for PubMedID 9005848

  • Stark spectroscopy: Applications in chemistry, biology, and materials science ANNUAL REVIEW OF PHYSICAL CHEMISTRY Bublitz, G. U., Boxer, S. G. 1997; 48: 213-242

    Abstract

    Stark spectroscopy has been applied to a wide range of molecular systems and materials. A generally useful method for obtaining electronic and vibrational Stark spectra that does not require sophisticated equipment is described. By working with frozen glasses it is possible to study nearly any molecular system, including ions and proteins. Quantitative analysis of the spectra provides information on the change in dipole moment and polarizability associated with a transition. The change in dipole moment reflects the degree of charge separation for a transition, a quantity of interest to a variety of fields. The polarizability change describes the sensitivity of a transition to an electrostatic field such as that found in a protein or an ordered synthetic material. Applications to donor-acceptor polyenes, transition metal complexes (metal-to-ligand and metal-to-metal mixed valence transitions), and nonphotosynthetic biological systems are reviewed.

    View details for Web of Science ID A1997YB98900008

    View details for PubMedID 9348658

  • Two-photon excitation of 4'-hydroxymethyl-4,5',8-trimethylpsoralen PHOTOCHEMISTRY AND PHOTOBIOLOGY Oh, D. H., Stanley, R. J., Lin, M., Hoeffler, W. K., Boxer, S. G., Berns, M. W., BAUER, E. A. 1997; 65 (1): 91-95

    Abstract

    Psoralens are a class of pharmaceutical agents commonly used to treat several cutaneous disorders. When irradiated with a mode-locked titanium: sapphire (Ti:sapphire) laser tuned to 730 nm, an aqueous solution of 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) emits blue light. The emission spectrum is centered at 452 nm and is identical to that obtained by one-photon excitation with UVA excitation, and its magnitude depends quadratically on the intensity of laser excitation. These results suggest that two-photon excitation occurs to a potentially photochemically active state. To estimate the two-photon absorption cross section, it was first necessary to measure the emission quantum yield of HMT using 365 nm excitation at room temperature that resulted in a value of 0.045 +/- 0.007. The two-photon absorption cross section of HMT at 730 nm is therefore estimated to be 20 x 10(-50) cm4 s (20 Göppert-Mayer). The excited-state photophysics and photochemistry of psoralens suggest potential applications to cutaneous phototherapy in diseases such as psoriasis and dystrophic epidermolysis bullosa.

    View details for Web of Science ID A1997WD16800021

    View details for PubMedID 9066288

  • Architecture and function of membrane proteins in planar supported bilayers: A study with photosynthetic reaction centers BIOCHEMISTRY Salafsky, J., Groves, J. T., Boxer, S. G. 1996; 35 (47): 14773-14781

    Abstract

    We present a simple and convenient method for creating fluid supported bilayers which contain oriented and functional photosynthetic reaction centers (RCs). The supported bilayers are prepared by fusion of proteoliposomes with a glass surface. The proteoliposomes are prepared by spontaneous insertion of RCs into preformed small, unilamellar vesicles. The RCs in these vesicles are shown to be oriented with the cytochrome c binding surface on the outside and the H-subunit facing inside. Upon fusion to glass surfaces, the RCs remain functional and highly oriented, with the cytochrome c binding surface exposed to the bulk solution. The RCs in the supported bilayers are at a surface density of order 10(11) RCs/cm2. The quality of the supported lipid bilayer is characterized by epifluorescence microscopy and the long-range lateral mobility of the lipids by fluorescence recovery after photobleaching. We demonstrate that homogeneous, fluid bilayers can be prepared over large areas (e.g., 1 cm2) of clean glass surfaces. The lipids in these supported bilayers are laterally mobile, and their diffusion coefficient agrees with values obtained in other fluid bilayer systems. This fluidity is unaffected by the presence of RCs; however, the RCs bearing a site-specific fluorescent label are immobile, despite retaining their charge separation and cytochrome c binding properties. We speculate that this results from interactions between the globular domain of the H-subunit and the glass substrate. Because of the unique spectroscopic and functional signatures associated with intact RCs, this system is one of the best characterized examples of a transmembrane protein in a supported bilayer at a nonbiological interface.

    View details for Web of Science ID A1996VV23700014

    View details for PubMedID 8942639

  • Electrical manipulation of glycan phosphatidyl inositol tethered proteins in planar supported bilayers BIOPHYSICAL JOURNAL Groves, J. T., Wulfing, C., Boxer, S. G. 1996; 71 (5): 2716-2723

    Abstract

    Electric fields have been used to manipulate and concentrate glycan-phosphatidyl inositol (GPI)-tethered proteins in planar supported bilayers. Naturally GPI-linked CD48, along with engineered forms of I-Ek and B7-2, in which their transmembrane domains have been genetically replaced with the GPI linkage, were studied. The proteins were labeled with fluorescently tagged antibodies, allowing the electric field-induced behavior to be followed by epifluorescence microscopy. All three protein complexes were observed to migrate toward the cathode with the B7-2 and CD48, each tethered to the membrane by a single GPI linker, moving significantly faster than the I-Ek, which has two GPI linkers. Patterns scratched into the membrane function as barriers to lateral diffusion and were used to isolate the proteins into highly concentrated corrals. All field-induced concentration profiles were completely reversible, indicating that the supported bilayer provides a stable, fluid environment in which GPI-tethered proteins can be manipulated. The ability to electrically control the spatial distribution of membrane-tethered proteins provides new opportunities for the study of biological membranes and the development of membrane-based devices.

    View details for Web of Science ID A1996VQ52000042

    View details for PubMedID 8913608

  • Structural biology - Another green revolution NATURE Boxer, S. G. 1996; 383 (6600): 484-485

    View details for Web of Science ID A1996VL75500028

    View details for PubMedID 8849718

  • Ultra-fast excited state dynamics in green fluorescent protein: Multiple states and proton transfer PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chattoraj, M., King, B. A., Bublitz, G. U., Boxer, S. G. 1996; 93 (16): 8362-8367

    Abstract

    The green fluorescent protein (GFP) of the jellyfish Aequorea Victoria has attracted widespread interest since the discovery that its chromophore is generated by the autocatalytic, posttranslational cyclization and oxidation of a hexapeptide unit. This permits fusion of the DNA sequence of GFP with that of any protein whose expression or transport can then be readily monitored by sensitive fluorescence methods without the need to add exogenous fluorescent dyes. The excited state dynamics of GFP were studied following photo-excitation of each of its two strong absorption bands in the visible using fluorescence upconversion spectroscopy (about 100 fs time resolution). It is shown that excitation of the higher energy feature leads very rapidly to a form of the lower energy species, and that the excited state interconversion rate can be markedly slowed by replacing exchangeable protons with deuterons. This observation and others lead to a model in which the two visible absorption bands correspond to GFP in two ground-state conformations. These conformations can be slowly interconverted in the ground state, but the process is much faster in the excited state. The observed isotope effect suggests that the initial excited state process involves a proton transfer reaction that is followed by additional structural changes. These observations may help to rationalize and motivate mutations that alter the absorption properties and improve the photo stability of GFP.

    View details for Web of Science ID A1996VB32500039

    View details for PubMedID 8710876

  • Trans effects in nitric oxide binding to myoglobin cavity mutant H93G BIOCHEMISTRY Decatur, S. M., Franzen, S., DEPILLIS, G. D., Dyer, R. B., Woodruff, W. H., Boxer, S. G. 1996; 35 (15): 4939-4944

    Abstract

    When nitric oxide (NO) binds to heme proteins, it exerts a repulsive trans effect on the proximal ligand, resulting in weakening or rupture of the proximal ligand-iron bond. The general question of whether NO binding generates a five-coordinate complex with proximal ligand release is important for the function of enzymes such as guanylate cyclase. This question can be addressed by studying NO binding to the myoglobin cavity mutant H93G, where the proximal histidine has been replaced by glycine. When this protein is expressed in the presence of imidazole (Im), an imidazole molecule occupies the proximal cavity and serves as a ligand to the iron [Barrick, D. (1994) Biochemistry 33, 6546-6554]. This proximal imidazole can be exchanged for a variety of exogenous ligands [DePillis, G.D., Decatur, S. M., Barrick, D., & Boxer, S.G. (1994) J. Am. Chem. Soc. 116, 6981-6982]. While CO binds to H93G(Im) to form a stable six-coordinate complex similar to that of the wild type and NO binds to wild-type myoglobin to form a six-coordinate complex, we find that the binding of NO to H93G(Im) under similar conditions results in the cleavage of the exogenous imidazole-iron bond at neutral pH, leaving a five-coordinate heme-NO complex, H93G-NO, inside the protein. When a large excess of imidazole is added to this five-coordinate NO complex, a six-coordinate complex can be formed; thus, the binding constant of a sixth ligand to the five-coordinate H93G-NO complex can be measured. This is found to be several orders of magnitude smaller than the binding constant of Im to the carbonmonoxy, deoxy, or the metcyano forms of protein. By replacement of Im with methyl-substituted imidazoles which have hindered or strained binding conformations, this binding constant can be reduced further and some of the factors responsible for favoring the five-coordinate form can be elucidated. Thus, the cavity mutant H93G provides a novel model system for studying the factors that control the coordination state of NO complexes of heme proteins and serves as a bridge between synthetic heme model complexes in simple solvents and site-directed mutants in the structured environment found in proteins.

    View details for Web of Science ID A1996UF52100034

    View details for PubMedID 8664286

  • Modulation of protein function by exogenous ligands in protein cavities: CO binding to a myoglobin cavity mutant containing unnatural proximal ligands BIOCHEMISTRY Decatur, S. M., DEPILLIS, G. D., Boxer, S. G. 1996; 35 (13): 3925-3932

    Abstract

    A variety of heterocyclic ligands can be exchanged into the proximal cavity of sperm whale myoglobin mutant H93G, providing a simple method for introduction of the equivalent of unnatural amino acid side chains into a functionally critical location in this protein. These modified proteins bind CO on the distal side. 1H NMR data on H93G(Im)CO, where Im is imidazole, demonstrate that the structure of the distal heme pocket in H93G(Im)CO is very similar to that of wild type; thus, the effects of the proximal ligand's properties on CO binding can be studied with minimal perturbation of distal pocket structure. The exogenous proximal ligands used in this study include imidazole (Im), 4-methylimidazole (4-MeIm), 4-bromoimidazole (4-BrIm), N-methylimidazole (N-MeIm), pyridine (Pyr), and 3-fluoropyridine (3-FPyr). Substitution of the proximal ligand is found to produce substantial changes in the CO on and off rates, the equilibrium binding constant, and the vibrational stretch frequency of CO. Many of the changes are as large as those reported for distal pocket mutants prepared by site-directed mutagenesis. The ability to systematically vary the nature of the proximal ligand is exploited to test the effects of particular properties of the proximal ligand on CO binding. For example, 4-MeIm and 4-BrIm are similar in size and shape but differ significantly in pKa. The same relationship is true for Pyr and 3-FPyr. By comparison of the IR spectra and CO recombination kinetics of these complexes, the effects of proximal ligand pKa on the CO binding are assessed. Likewise, N-MeIm and 4-MeIm are similar in size and pKa but differ in their ability to hydrogen bond to amino acid residues in the proximal cavity. Comparisons of IR spectra and CO binding kinetics in these complexes reveal that proximal ligand conformation and hydrogen bonding affect the kinetics of CO binding. The mechanism of proximal ligand exchange between solution and the proximal cavity in CO complexes was investigated by obtaining the 19F NMR spectrum of H93G(3-FPyr)CO, whose 19F signal can be observed without interference from resonances of the protein. The proximal ligand is found to exchange within a few seconds by saturation transfer. This exchange rate is about 2 orders of magniture faster than what is observed for the isoelectronic metcyano complex [Decatur, S. M., & Boxer, S. G. (1995) Biochemistry 34, 2122-2129]; in both the ferrous CO and ferric cyano complexes, the proximal ligand exchange rate is independent of ligand concentration. These results suggest that the rate-limiting step in proximal ligand exchange is breakage of the iron-ligand bond, followed by rapid diffusion of the ligand through the protein to bulk solution.

    View details for Web of Science ID A1996UC97700008

    View details for PubMedID 8672423

  • Mg coordination by amino acid side chains is not required for assembly and function of the special pair in bacterial photosynthetic reaction centers BIOCHEMISTRY GOLDSMITH, J. O., King, B., Boxer, S. G. 1996; 35 (7): 2421-2428

    Abstract

    A conserved histidine serves as the axial ligand to the Mg of bacteriochlorophylls in the photosynthetic reaction center (RC) and many other photosynthetic systems. The histidine axial ligands to each and both bacteriochlorophylls of the special-pair primary electron donor of the Rhodobacter sphaeroides RC have been replaced with glycine to create a cavity. In each case, RCs assemble and a normal special-pair comprised of Mg-containing bacteriochlorophylls is formed, as judged by many different spectroscopic and functional probes (e.g., absorption and Stark spectra, *P decay kinetics, P+Q(A)- recombination rate, and the redox potential of P). In contrast with heme proteins, where this strategy has been exploited to introduce exogenous organic ligands that can greatly affect the functional properties of the protein [DePillis, G. D., Decatur, S. M., Barrick, D., & Boxer, S. G. (1994) J. Am. Chem. Soc. 116, 6981-6982], addition of exogenous imidazole, pyridine, and ethanethiol has no measurable effect on the functional properties of the special pair in these cavity mutants. FT-Raman spectroscopy is used to provide more detailed information on local interactions around the special pair. Data in the core-size marker mode and carbonyl stretching region suggest that an adventitious ligand replaces histidine as the axial ligand to bacteriochlorophylls in the cavity mutants. We speculate that this ligand is water. Furthermore, the position of the core-size marker mode changes when the cavity mutant RCs are incubated with exogenous ligands such as imidazole, pyridine, or ethanethiol, suggesting that the axial ligand to the special pair BChls can be exchanged in the cavity mutants. Interestingly the temperature dependence of P+Q(A)- recombination kinetics is very similar in the cavity mutants and WT, suggesting that the axial ligands to the special pair are not significant contributors to the solvent reorganization energy for this reaction. These results lead to the surprising conclusion that the nature of the axial ligand to the special pair has little influence on the properties of the macrocycle, and that axial coordination from the protein by histidine is not required for bacteriochlorophyll binding or for efficient electron transfer in the RC.

    View details for Web of Science ID A1996TW34500041

    View details for PubMedID 8652585

  • Electric field-induced concentration gradients in planar supported bilayers BIOPHYSICAL JOURNAL Groves, J. T., Boxer, S. G. 1995; 69 (5): 1972-1975

    Abstract

    A simple method of generating electric field-induced concentration gradients in planar supported bilayers has been developed. Gradients of charged, fluorescently labeled probes were visualized by epifluorescence microscopy and could be observed at field strengths as low as 1 V/cm. Steady-state concentration gradients can be described by a simple competition between random diffusion and electric field-induced drift. A model based on this principle has been used to determine the diffusion coefficient of the fluorescent probes. This technique achieves a degree of electrical manipulation of supported bilayers that offers a variety of possibilities for the development of new molecular architectures and the study of biological membranes.

    View details for Web of Science ID A1995TV01700034

    View details for PubMedID 8580340

  • A TEST OF THE ROLE OF ELECTROSTATIC INTERACTIONS IN DETERMINING THE CO STRETCH FREQUENCY IN CARBONMONOXYMYOGLOBIN BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Decatur, S. M., Boxer, S. G. 1995; 212 (1): 159-164

    Abstract

    The vibrational frequency of CO bound to myoglobin can be varied by up to 60 cm-1 by making site-specific mutations in the distal pocket. These changes may result from specific chemical interactions between distal amino acids and the CO or from changes in the electrostatic field of the distal pocket. In this paper, we separate the relative contributions of these two effects by comparing the IR spectra of the carbonmonoxy complexes of human myoglobin mutants V68N, V68D, and V68E. The effect of replacing valine with these polar amino acids on the electrostatic environment of the distal heme pocket has been independently determined earlier by measurements of the heme reduction potential and electronic absorption spectral band shifts. While all three mutations result in a negative dipole pointing towards the CO ligand, the CO stretch frequency shifts differently in each case. These differences are attributed to specific chemical interactions between the amino acids and the CO ligand.

    View details for Web of Science ID A1995RG81500024

    View details for PubMedID 7612000

  • STARK-EFFECT SPECTROSCOPY OF TRYPTOPHAN BIOPHYSICAL JOURNAL Pierce, D. W., Boxer, S. G. 1995; 68 (4): 1583-1591

    Abstract

    The change in permanent dipole moment (magnitude of delta mu) for the transition from the 1La state to the ground state of tryptophan is the key photophysical parameter for the interpretation of tryptophan fluorescence spectra in terms of static and dynamic dielectric properties of the surrounding medium. We report measurement of this parameter by means of electric field effect (Stark) spectroscopy for N-acetyl-L-tryptophanamide (NATA) in two solvents, the single tryptophan containing peptide melittin, and 5-methoxytryptophan. The values ranged from 5.9 to 6.2 +/- 0.4 Debye/f for NATA and melittin, where f represents the local field correction. The 1Lb magnitude of delta mu was much smaller. Application of Stark spectroscopy to these chromophores required decomposition of the near-UV absorption into the 1La and 1Lb bands by measurement of the fluorescence excitation anisotropy spectrum and represents an extension of the method to systems where band overlap would normally preclude quantitative analysis of the Stark spectrum. The results obtained for 5-methoxytryptophan point out limitations of this method of spectral decomposition. The relevance of these results to the interpretation of steady-state and time-resolved spectroscopy of tryptophan is discussed.

    View details for Web of Science ID A1995RD16300036

    View details for PubMedID 7787044

  • H-1-NMR CHARACTERIZATION OF MYOGLOBINS WHERE EXOGENOUS LIGANDS REPLACE THE PROXIMAL HISTIDINE BIOCHEMISTRY Decatur, S. M., Boxer, S. G. 1995; 34 (7): 2122-2129

    Abstract

    The role of the proximal ligand in determining the structure and ligand binding properties of sperm whale myoglobin has been investigated using the mutant H93G(L), where the proximal histidine has been replaced with glycine, creating a cavity which can be occupied by a variety of exogenous ligands, L, to the iron [Barrick, D. (1994) Biochemistry 33, 6546-6554; DePillis, G.D., Decatur, S.M., Barrick, D., & Boxer, S.G. (1994) J. Am. Chem. Soc. 116, 6981-6982]. In this report, we present the assignments of selected protons of the heme and heme pocket residues in the metcyano complexes of H93G with Im and a series of methyl-substituted Ims [H93G(Im)CN, H93G(N-MeIm)CN, H93G(2-MeIm)CN, H93G-(4-MeIm)CN]. Each complex has a unique 1H NMR spectrum, providing a fingerprint for documenting the ligand exchange phenomenon. Moreover, the identification of NOEs between the protons of proximal ligands and protons of proximal pocket amino acid residues confirms that the new ligand occupies the proximal cavity in solution. The pattern of hyperfine-shifted heme methyl resonances in H93G(Im)CN is very different from that of wild-type Mb, consistent with the differences compared to wild-type in is very different from that of wild-type Mb, consistent with the differences compared to wild-type in orientation of the proximal imidazole observed in the X-ray crystal structure of H93G(Im) [Barrick, D. (1994) Biochemistry 33, 6546-6554]. Addition of deuterated Im to H93G(Im)CN permits direct observation of exchange of proximal ligands with ligands from solution; exchange of Im for deuterated Im in the metcyano complex occurs with half-life of around 10 min.(ABSTRACT TRUNCATED AT 250 WORDS)

    View details for Web of Science ID A1995QH59000004

    View details for PubMedID 7857922

  • FUNCTIONAL-ASPECTS OF ULTRA-RAPID HEME DOMING IN HEMOGLOBIN, MYOGLOBIN, AND THE MYOGLOBIN MUTANT H93G JOURNAL OF BIOLOGICAL CHEMISTRY Franzen, S., Bohn, B., Poyart, C., DEPILLIS, G., Boxer, S. G., Martin, J. L. 1995; 270 (4): 1718-1720

    Abstract

    Heme iron out-of-plane displacement following ligand dissociation in hemoglobin, myoglobin, and the proximal cavity mutant H93G is shown to be as rapid as the heme iron out-of-plane vibrational period by sub-picosecond time-resolved resonance Raman spectroscopy. The results demonstrate that the effect of steric repulsion initiated by the spin change of the iron gives rise to heme doming independent of covalent attachment of the proximal ligand to the protein. It is concluded that the protein plays a passive role in the initial ultrafast heme iron motion toward the out-of-plane position observed in the deoxy structure of hemoglobin and myoglobin. The results suggest that the spin change of the heme iron is the primary cause of rapid heme doming and that steric repulsion of the proximal ligand with the heme plays a secondary role in forcing the iron out of the heme plane.

    View details for Web of Science ID A1995QD20400038

    View details for PubMedID 7829506

  • SPECTROSCOPIC STUDY OF SER92 MUTANTS OF HUMAN MYOGLOBIN - HYDROGEN-BONDING EFFECT OF SER92 TO PROXIMAL HIS93 ON STRUCTURE AND PROPERTY OF MYOGLOBIN BIOCHEMISTRY Shiro, Y., Iizuka, T., Marubayashi, K., Ogura, T., Kitagawa, T., Balasubramanian, S., Boxer, S. G. 1994; 33 (50): 14986-14992

    Abstract

    Neutron diffraction studies have demonstrated that the hydroxyl group oxygen of Ser92(F7) is hydrogen bonded to the proximal His93(48) N epsilon H proton in myoglobin (Mb) [Cheng, X., & Shoenborn, B. P. (1991) J. Mol. Biol. 220, 381-399]. In order to examine the importance of this hydrogen bond, Ser92 was replaced with Ala and Asp in human Mb. By comparing the optical, 1H-NMR, resonance Raman, and IR spectra of Mb(S92A) in several spin and oxidation states with those of wild-type Mb, it was found that the mutation causes a structural change on the heme proximal side but not on the distal side. Comparison of the NMR spectra of the cyanomet form of Mb(S92A) and Mb(WT) suggests that the imidazole plane of His93 rotates somewhat around the Fe-N delta (His93) bond upon loss of the hydrogen bond between His93 and Ser92. The 2D 1H-NMR measurements of the CO complexes show that mutation of Ser92 to Ala changes the relative position of the His97 imidazole group to the heme plane, but the change is not so drastic as reported in the crystal data of Ser92 mutant of pig Mb [Smerdon et al. (1993) Biochemistry 32, 5132-5138]. On the other hand, ligand (CO, O2) binding is only slightly affected by this mutation. From these results, we conclude that the Ser92-His93 hydrogen bond maintains the protein structure of the proximal heme pocket, but it does not strongly affect the electronic structure of the heme as well as of the His93 imidazole ring.(ABSTRACT TRUNCATED AT 250 WORDS)

    View details for Web of Science ID A1994PY28700006

    View details for PubMedID 7999755

  • CASTING A COLD EYE OVER MYOGLOBIN NATURE STRUCTURAL BIOLOGY Boxer, S. G., AUFINRUD, P. A. 1994; 1 (11): 749-751

    View details for Web of Science ID A1994QN70800002

    View details for PubMedID 7634080

  • DETERMINATION OF THE CARBON-MONOXIDE BINDING CONSTANTS OF MYOGLOBIN MUTANTS - COMPARISON OF KINETIC AND EQUILIBRIUM METHODS BIOCHEMISTRY Balasubramanian, S., Lambright, D. G., Simmons, J. H., Gill, S. J., Boxer, S. G. 1994; 33 (27): 8355-8360

    Abstract

    The carbon monoxide (CO) binding constants of human myoglobin (Mb) and several single-site mutants have been determined using two different methods. In the kinetic method, which is commonly used for this ligand, the overall association (k(on)) and dissociation (k(off)) rates of CO were measured by flash photolysis and NO replacement, respectively, and the ratio k(on)/k(off) was calculated. In the equilibrium method, the binding constant Keq was measured directly using a thin-layer technique. These two measurements yield similar results for human wild-type Mb but differ significantly for some of the mutants. Possible reasons for these discrepancies are analyzed. A model assuming the presence of interconverting conformers with different association and dissociation rates is considered in light of infrared measurements on the CO stretching frequency in the MbCO forms of the same proteins [Balasubramanian et al. (1993a) Proc. Natl. Acad. Sci, U.S.A. 90, 4718]. It is suggested that in the case of some mutants which exhibit multiple conformations, this model may lead to nonequilibrium kinetics, which could produce the observed discrepancies between the kinetic and equilibrium determinations of the binding constant. These results suggest that both equilibrium and kinetic data should be obtained, even for a monomeric protein such as Mb, before the relative stabilities of mutants can be meaningfully compared.

    View details for Web of Science ID A1994NX32900024

    View details for PubMedID 8031769

  • NEAR-INFRARED RESONANCE RAMAN-SPECTROSCOPY OF THE SPECIAL PAIR AND THE ACCESSORY BACTERIOCHLOROPHYLLS IN PHOTOSYNTHETIC REACTION CENTERS JOURNAL OF PHYSICAL CHEMISTRY Cherepy, N. J., Shreve, A. P., Moore, L. J., Franzen, S., Boxer, S. G., Mathies, R. A. 1994; 98 (23): 6023-6029
  • ANATOMY AND DYNAMICS OF A LIGAND-BINDING PATHWAY IN MYOGLOBIN - THE ROLES OF RESIDUE-45, RESIDUE-60, RESIDUE-64, AND RESIDUE-68 BIOCHEMISTRY Lambright, D. G., Balasubramanian, S., Decatur, S. M., Boxer, S. G. 1994; 33 (18): 5518-5525

    Abstract

    In order for diatomic ligands to enter and exit myoglobin, there must be substantial displacements of amino acid side chains from their positions in the static X-ray structure. One pathway, involving Arg/Lys45, His64, and Val68, has been studied in greatest detail. In an earlier study (Lambright et al., 1989) we reported the surprising result that mutation of the surface residue Lys45 to arginine lowers the inner barrier to CO rebinding. Until then, it had been thought that this barrier primarily involves interior distal pocket residues such as His64 and Val68. In this report, we present a detailed study of the CO rebinding kinetics in aqueous solution of a series of single- and double-site mutants of human myoglobin at positions 64, 68, 45, and 60. On the basis of the observed kinetics, we propose that the effect of surface residue 45 on the inner barrier can be explained by a chain of interactions between surface and pocket residues. Very large, and in some cases unexpected, changes are observed in the kinetics of recombination and in the partitioning between geminate and bimolecular recombination.

    View details for Web of Science ID A1994NK97300021

    View details for PubMedID 8180174

  • ULTRAFAST MEASUREMENTS OF GEMINATE RECOMBINATION OF NO WITH SITE-SPECIFIC MUTANTS OF HUMAN MYOGLOBIN JOURNAL OF MOLECULAR BIOLOGY Petrich, J. W., Lambry, J. C., Balasubramanian, S., Lambright, D. G., Boxer, S. G., Martin, J. L. 1994; 238 (3): 437-444

    Abstract

    Flash photolysis studies of NO recombination to heme proteins offer a direct probe of protein structural changes on the tens of picoseconds timescale where they can be compared with molecular dynamics simulations. The geminate recombination of NO to site-specific mutants of human myoglobin (Mb) was studied following photodissociation of the MbNO form. Single amino acid changes were introduced at positions Val68, His64, Lys45 and Asp60 because motions of residues at these positions are generally regarded as important for the mechanism of ligand binding. In sharp contrast to the properties of simple porphyrin-NO complexes, the rebinding kinetics are found to be non-exponential for all mutants, even in aqueous solution at 298 K. The Val68 and His64 mutants substantially affect the NO rebinding rates but, surprisingly, so do changes on the protein surface that are further away from the iron. These changes in kinetics occur on a tens of picoseconds timescale, and therefore there is either a fast communication between protein residues over quite long distances or there are subtle differences in protein structure that exert great control over the reaction dynamics. Various models for the rebinding kinetics are evaluated. A model-free approach to data analysis using the maximum entropy method is found to be most useful. This analysis shows that the rate distributions are very different for the mutants, but are generally bimodal.

    View details for Web of Science ID A1994NK95500011

    View details for PubMedID 8176734

  • DIELECTRIC ASYMMETRY IN THE PHOTOSYNTHETIC REACTION-CENTER SCIENCE Steffen, M. A., Lao, K. Q., Boxer, S. G. 1994; 264 (5160): 810-816

    Abstract

    Although the three-dimensional structure of the bacterial photosynthetic reaction center (RC) reveals a high level of structural symmetry, with two nearly equivalent potential electron transfer pathways, the RC is functionally asymmetric: Electron transfer occurs along only one of the two possible pathways. In order to determine the origins of this symmetry breaking, the internal electric field present in the RC when charge is separated onto structurally characterized sites was probed by using absorption band shifts of the chromophores within the RC. The sensitivity of each probe chromophore to an electric field was calibrated by measuring the Stark effect spectrum, the change in absorption due to an externally applied electric field. A quantitative comparison of the observed absorption band shifts and those predicted from vacuum electrostatics gives information on the effective dielectric constant of the protein complex. These results reveal a significant asymmetry in the effective dielectric strength of the protein complex along the two potential electron transfer pathways, with a substantially higher dielectric strength along the functional pathway. This dielectric asymmetry could be a dominant factor in determining the functional asymmetry of electron transfer in the RC.

    View details for Web of Science ID A1994NJ94900023

    View details for PubMedID 17794722

  • DISCOVERY OF NEW LIGAND-BINDING PATHWAYS IN MYOGLOBIN BY RANDOM MUTAGENESIS NATURE STRUCTURAL BIOLOGY Huang, X. H., Boxer, S. G. 1994; 1 (4): 226-229

    Abstract

    A random library of single amino acid mutants of myoglobin was generated using a highly efficient, single-base-misincorporation random mutagenesis method to discover new ligand-binding pathways in myoglobin. A surprisingly large fraction of the library exhibits ligand-binding kinetics that are substantially different from the wild-type protein. In addition to residues 45, 64 and 68, which comprise the best studied ligand-binding pathway single mutations of several other clusters of residues far away from that pathway are discovered which profoundly affect the ligand-binding kinetics. These results provide a new approach to explore the relationship between the fluctuations in protein structure and function.

    View details for Web of Science ID A1994NU32300011

    View details for PubMedID 7656050

  • DYNAMICS OF PROTEIN RELAXATION IN SITE-SPECIFIC MUTANTS OF HUMAN MYOGLOBIN BIOCHEMISTRY Lambright, D. G., Balasubramanian, S., Boxer, S. G. 1993; 32 (38): 10116-10124

    Abstract

    We have recently reported spectroscopic evidence for structural relaxation of myoglobin (Mb) following photodissociation of MbCO [Lambright, D. G., Balasubramanian, S., & Boxer, S. G. (1991) Chem. Phys. 158, 249-260]. In this paper we report measurements for a series of single amino acid mutants of human myoglobin on the distal side of the heme pocket (positions 45, 64, and 68) in order to examine specific structural determinants involved in this conformational relaxation and to determine the nature of the coupling between relaxation and the functional process of ligand binding. The kinetics of ligand binding and conformational relaxation were monitored by transient absorption spectroscopy in the Soret spectral region, and the results are analyzed using a four-state ligand binding model. Two principal results emerge: (1) amino acid substitutions in the distal heme pocket affect the kinetics of the nonequilibrium conformational relaxation and (2) the rate of ligand escape from the protein matrix is not significantly perturbed by the distal heme pocket mutations.

    View details for Web of Science ID A1993LZ63800030

    View details for PubMedID 8399137

  • PERTURBATIONS OF THE DISTAL HEME POCKET IN HUMAN MYOGLOBIN MUTANTS PROBED BY INFRARED-SPECTROSCOPY OF BOUND CO - CORRELATION WITH LIGAND-BINDING KINETICS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Balasubramanian, S., Lambright, D. G., Boxer, S. G. 1993; 90 (10): 4718-4722

    Abstract

    The infrared spectra of CO bound to human myoglobin and myoglobin mutants at positions His-64, Val-68, Asp-60, and Lys-45 on the distal side have been measured between 100 and 300 K. Large differences are observed with mutations at His-64 and Val-68 as well as with temperature and pH. Although distal His-64 is found to affect CO bonding, Val-68 also plays a major role. The variations are analyzed qualitatively in terms of a simple model involving steric interaction between the bound CO and the distal residues. A strong correlation is found between the final barrier height to CO recombination and the CO stretch frequency: as compared to wild type, the barrier is smaller in those mutants that have a higher CO stretch frequency (vCO) and vice versa. Possible reasons for this correlation are discussed. It is emphasized that the temperature and pH dependence of both the kinetics and the infrared spectra must be measured to obtain a consistent picture.

    View details for Web of Science ID A1993LC72000083

    View details for PubMedID 8506324

  • CO RECOMBINATION TO HUMAN MYOGLOBIN MUTANTS IN GLYCEROL WATER SOLUTIONS BIOCHEMISTRY Balasubramanian, S., Lambright, D. G., Marden, M. C., Boxer, S. G. 1993; 32 (9): 2202-2212

    Abstract

    The kinetics of CO recombination to site-specific mutants of human myoglobin have been studied by flash photolysis in the temperature range 250-320 K on the nanosecond to second time scale in 75% glycerol at pH 7. The mutants were constructed to examine specific proposals concerning the roles of Lys 45, Asp 60, and Val 68 in the ligand binding process. It is found that ligand recombination is nonexponential for all the mutants and that both the geminate amplitude and rate show large variations. The results are interpreted in terms of specific models connecting the dynamics and structure. It is shown that removal of the charged group at position 45 does not substantially affect the barrier height for escape or entry of the ligand; therefore the breakage of the salt bridge linking Lys 45, Asp 60, and a heme propionate is ruled out as the rate-determining barrier for this process. On the other hand, it is found that the escape barrier decreases roughly as size of the residue at position 68 increases, in the order Ala > Val > Asn > Leu. The residue at position 68 is also a major contributor to the final barrier to rebinding, but the barrier height shows no correlation with residue size and is more dependent on the stereochemistry of the residue. A molecular mechanism for ligand binding that is consistent with the results is discussed, and supporting evidence for this mechanism is examined.

    View details for Web of Science ID A1993KQ86000011

    View details for PubMedID 8443162

  • SPECTROSCOPIC AND REDOX PROPERTIES OF SYM1 AND (M)F195H - RHODOBACTER-CAPSULATUS REACTION CENTER SYMMETRY MUTANTS WHICH AFFECT THE INITIAL ELECTRON-DONOR BIOCHEMISTRY Stocker, J. W., Taguchi, A. K., MURCHISON, H. A., Woodbury, N. W., Boxer, S. G. 1992; 31 (42): 10356-10362

    Abstract

    The redox properties, absorption, electroabsorption, CD, EPR, and P+QA- recombination kinetics have been measured for the special pairs of two mutants of Rhodobacter capsulatus reaction centers involving amino acid changes in the vicinity of the special pair, P. Both mutants symmetrize amino acid residues so that portions of the M-sequence are replaced with L-sequence: sym1 symmetrizes all residues between M187 and M203, whereas (M)F195H is a single amino acid subset of the sym1 mutation. (M)F195H introduces a His residue in a position where it is likely to form a hydrogen bond to the acetyl group of the M-side bacteriochlorophyll of P. For both mutants compared with wild-type, (i) the redox potential is at least 100 meV greater, (ii) the P+QA- recombination rate is about twice as fast at room temperature, and (iii) the large electroabsorption feature for the QY band of P is shifted relative to the absorption spectrum. The comparison of the properties observed for the sym1 and (M)F195H reaction center mutants and the differences between these mutants and wild-type suggest that residue M195 is an important determinant of the properties of the special pair.

    View details for Web of Science ID A1992JV25800025

    View details for PubMedID 1329946

  • BIOCHEMICAL-CHARACTERIZATION AND ELECTRON-TRANSFER REACTIONS OF SYM1, A RHODOBACTER-CAPSULATUS REACTION CENTER SYMMETRY MUTANT WHICH AFFECTS THE INITIAL ELECTRON-DONOR BIOCHEMISTRY Taguchi, A. K., Stocker, J. W., ALDEN, R. G., Causgrove, T. P., Peloquin, J. M., Boxer, S. G., Woodbury, N. W. 1992; 31 (42): 10345-10355

    Abstract

    A 51 bp section of the Rhodobacter capsulatus photosynthetic reaction center M subunit gene (nucleotides M562-M612 of the pufM structural sequence) encoding amino acids M187-M203 was replaced by the homologous region of the L subunit gene. This resulted in the symmetrization of much of the amino acid environment of the reaction center initial electron donor, P. This is the first in a series of large-scale symmetry mutations and is referred to as sym1. The sym1 mutant was able to grow photosynthetically, indicating that reaction center function was largely intact. Isolated reaction centers showed an approximately 10-nm blue shift in the QY band of P. The standard free energy change between P* and P+BphA- determined from analysis of the long-lived fluorescence from quinone-reduced reaction centers decreased from about -120 meV in the wild-type to about -75 meV in the sym1 mutant. A 65-70% quantum yield of electron transfer from P* to P+QA- was observed, most of the yield loss occurring between P* and P+BphA-. The decay of the stimulated emission from P* was about 3-fold slower in this mutant than in the wild-type. Time-resolved spectral analysis of the charge-separated intermediates formed in sym1 reaction centers indicated that the major product was P+BphA-. A model-dependent analysis of the observed rates and electron-transfer yields gave the following microscopic rate constants for sym1 reaction centers (wild-type values under the same conditions are given in parentheses): [formula: see text] Analysis of the sym1 mutant, mutants near P made by other groups, and interspecies variation of amino acids in the vicinity of P suggests that the protein asymmetry in the environment of the initial electron donor is important for optimizing the rate and yield of electron transfer, but is not strictly required for overall reaction center function.

    View details for Web of Science ID A1992JV25800024

    View details for PubMedID 1420154

  • RAPID-FLOW RESONANCE RAMAN-SPECTROSCOPY OF BACTERIAL PHOTOSYNTHETIC REACTION CENTERS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Shreve, A. P., Cherepy, N. J., Franzen, S., Boxer, S. G., Mathies, R. A. 1991; 88 (24): 11207-11211

    Abstract

    Rapid-flow resonance Raman vibrational spectra of bacterial photosynthetic reaction centers from the R-26 mutant of Rhodobacter sphaeroides have been obtained by using excitation wavelengths (810-910 nm) resonant with the lowest energy, photochemically active electronic absorption. The technique of shifted excitation Raman difference spectroscopy is used to identify genuine Raman scattering bands in the presence of a large fluorescence background. The comparison of spectra obtained from untreated reaction centers and from reaction centers treated with the oxidant K3Fe(CN)6 demonstrates that resonance enhancement is obtained from the special pair. Relatively strong Raman scattering is observed for special pair vibrations with frequencies of 36, 94, 127, 202, 730, and 898 cm-1; other modes are observed at 71, 337, and 685 cm-1. Qualitative Raman excitation profiles are reported for some of the strong modes, and resonance enhancement is observed to occur throughout the near-IR absorption band of the special pair. These Raman data determine which vibrations are coupled to the optical absorption in the special pair and, thus, probe the nuclear motion that occurs after electronic excitation. Implications for the interpretation of previous hole-burning experiments and for the excited-state dynamics and photochemistry of reaction centers are discussed.

    View details for Web of Science ID A1991GV87700047

    View details for PubMedID 1763034

  • STARK-EFFECT SPECTROSCOPY OF CAROTENOIDS IN PHOTOSYNTHETIC ANTENNA AND REACTION CENTER COMPLEXES BIOCHIMICA ET BIOPHYSICA ACTA Gottfried, D. S., Steffen, M. A., Boxer, S. G. 1991; 1059 (1): 76-90

    Abstract

    The effects of electric fields on the absorption spectra of the carotenoids spheroidene and spheroidenone in photosynthetic antenna and reaction center complexes (wild-type and several mutants) from purple non-sulfur bacteria are compared with those for the isolated pigments in organic glasses. In general, the field effects are substantially larger for the carotenoid in the protein complexes than for the extracted pigments and larger for spheroidenone than spheroidene. Furthermore, the electrochromic effects for carotenoids in all complexes are much larger than those for the Qx transitions of the bacteriochlorophyll and bacteriopheophytin pigments which absorb in the 450-700 nm spectral region. The underlying mechanism responsible for the Stark effect spectra in the complexes is found to be dominated by a change in permanent dipole moment of the carotenoid upon excitation. The magnitude of this dipole moment change is found to be considerably larger in the B800-850 complex compared to the reaction center for spheroidene; it is approximately equivalent in the two complexes for spheroidenone. These results are discussed in terms of the effects of differences in the carotenoid functional groups, isomers and perturbations on the electronic structure from interactions with the organized environment in the proteins. these data provide a quantitative basis for the analysis of carotenoid bandshifts which are used to measure transmembrane potential, and they highlight some of the pitfalls in making such measurements on complex membranes containing multiple populations of carotenoids. The results for spheroidenone should be useful for studies of mutant proteins, since mutant strains are often grown semi-aerobically to minimize reversion.

    View details for Web of Science ID A1991GC28300008

    View details for PubMedID 1873299

  • LARGE PROTEIN-INDUCED DIPOLES FOR A SYMMETRICAL CAROTENOID IN A PHOTOSYNTHETIC ANTENNA COMPLEX SCIENCE Gottfried, D. S., Steffen, M. A., Boxer, S. G. 1991; 251 (4994): 662-665

    Abstract

    Unusually large electric field effects have been measured for the absorption spectra of carotenoids (spheroidene) in the B800-850 light-harvesting complex from the photosynthetic bacterium Rhodobacter sphaeroides. Quantitative analysis shows that the difference in the permanent dipole moment between the ground state and excited states in this protein complex is substantially larger than for pure spheroidene extracted from the protein. The results demonstrate the presence of a large perturbation on the electronic structure of this nearly symmetric carotenoid due to the organized environment in the protein. This work also provides an explanation for the seemingly anomalous dependence of carotenoid band shifts on transmembrane potential and a generally useful approach for calibrating electric field-sensitive dyes that are widely used to probe potentials in biological systems.

    View details for Web of Science ID A1991EW39900042

    View details for PubMedID 1992518

  • ELECTRIC-FIELD EFFECTS ON THE INITIAL ELECTRON-TRANSFER KINETICS IN BACTERIAL PHOTOSYNTHETIC REACTION CENTERS JOURNAL OF PHYSICAL CHEMISTRY Lockhart, D. J., Kirmaier, C., HOLTEN, D., Boxer, S. G. 1990; 94 (18): 6987-6995
  • STARK SPECTROSCOPY OF THE RHODOBACTER-SPHAEROIDES REACTION CENTER HETERODIMER MUTANT PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA HAMMES, S. L., Mazzola, L., Boxer, S. G., GAUL, D. F., Schenck, C. C. 1990; 87 (15): 5682-5686

    Abstract

    The effect of an electric field has been measured on the absorption spectrum (Stark effect) of the heterodimer mutant (M)H202L of Rhodobacter sphaeroides reaction centers, where the primary electron donor consists of one bacteriochlorophyll alpha and one bacteriopheophytin alpha. The electronic absorption spectrum of the heterodimer mutant from 820-950 nm is relatively featureless in a poly(vinyl alcohol) film, but it exhibits some structure in a glycerol/water glass at 77 K. A feature is seen in the Stark effect spectrum of the heterodimer at 77 K centered at 927 and 936 nm in poly(vinyl alcohol) and a glycerol/water glass, respectively. This feature has approximately the same shape and width as the Stark effect for the primary electron donor of the wild type, which consists of a pair of bacteriochlorophyll alpha molecules. The angle zeta A between the transition moment at the frequency of absorption and the difference dipole delta muA is 36 +/- 2 degrees in the wild type and 32 +/- 2 degrees for that feature in the heterodimer. A range of values for [delta muA] = (13-17)/f Debye units (where f is the local field correction) is obtained for the 936-nm feature in glycerol/water, depending on analysis method. This feature is interpreted as arising from a transition to the lower exciton state of the heterodimer, which is more strongly mixed with a low-lying charge transfer transition than in the wild type.

    View details for Web of Science ID A1990DR71400020

    View details for PubMedID 2198569

  • X-RAY CRYSTAL-STRUCTURE OF A RECOMBINANT HUMAN MYOGLOBIN MUTANT AT 2.8 A RESOLUTION JOURNAL OF MOLECULAR BIOLOGY Hubbard, S. R., Hendrickson, W. A., Lambright, D. G., Boxer, S. G. 1990; 213 (2): 215-218

    Abstract

    We have grown crystals in trigonal space group P3(2)21 of a mutant human myoglobin, aquomet form, in which lysine at position 45 has been replaced by arginine and cysteine at position 110 has been replaced by alanine. Suitable crystals of native recombinant human myoglobin have not been obtained. We have used the molecular replacement method to determine the X-ray crystal structure of the mutant at 2.8 A resolution. At the present stage of refinement, the crystallographic R-value for the model, with tightly restrained stereochemistry, is 0.158 for 5.0 to 2.8 A data. As expected, the overall structure is quite similar to the sperm whale myoglobin structure. Arginine 45 adopts a well-ordered conformation similar to that found in aquomet sperm whale myoglobin.

    View details for Web of Science ID A1990DE23600005

    View details for PubMedID 2342104

  • CHARACTERIZATION OF THE OVERALL AND INTERNAL DYNAMICS OF SHORT OLIGONUCLEOTIDES BY DEPOLARIZED DYNAMIC LIGHT-SCATTERING AND NMR RELAXATION MEASUREMENTS BIOCHEMISTRY Eimer, W., Williamson, J. R., Boxer, S. G., Pecora, R. 1990; 29 (3): 799-811

    Abstract

    The dynamics of three synthetic oligonucleotides d(CG)4, d(CG)6, and d(CGCGTTGTTCGCG) of different length and shape were studied in solution by depolarized dynamic light scattering (DDLS) and time-resolved nuclear Overhauser effect cross-relaxation measurements. For cylindrically symmetric molecules the DDLS spectrum is dominated by the rotation of the main symmetry axis of the cylinder. The experimental correlation times describe the rotation of the oligonucleotides under hydrodynamic stick boundary conditions. It is shown that the hydrodynamic theory of Tirado and Garcia de la Torre gives good predictions of the rotational diffusion coefficients of cylindrically symmetric molecules of the small axial ratios studied here. These relations are used to calculate the solution dimensions of the DNA fragments from measured correlation times. The hydrodynamic diameter of the octamer and dodecamer is 20.5 +/- 1.0 A, assuming a rise per base of 3.4 A. The tridecamer, d(CGCGTTGTTCGCG), adopts a hairpin structure with nearly spherical dimensions and a diameter of 23.0 +/- 2.0 A. The DDLS relaxation measurements provide a powerful method for distinguishing between different conformations of the oligonucleotides (e.g., DNA double-helix versus hairpin structure). Furthermore, the rotational correlation times are a very sensitive probe of the length of different fragments. The NMR results reflect the anisotropic motion of the molecules as well as the amount of local internal motion present. The experimental correlation time from NMR is determined by the rotation of both the short and long axes of the oligonucleotide.(ABSTRACT TRUNCATED AT 250 WORDS)

    View details for Web of Science ID A1990CK38000030

    View details for PubMedID 2337597

  • MECHANISMS OF LONG-DISTANCE ELECTRON-TRANSFER IN PROTEINS - LESSONS FROM PHOTOSYNTHETIC REACTION CENTERS ANNUAL REVIEW OF BIOPHYSICS AND BIOPHYSICAL CHEMISTRY Boxer, S. G. 1990; 19: 267-299

    View details for Web of Science ID A1990DJ35500012

    View details for PubMedID 2194478

  • LIGAND AND PROTON-EXCHANGE DYNAMICS IN RECOMBINANT HUMAN MYOGLOBIN MUTANTS JOURNAL OF MOLECULAR BIOLOGY Lambright, D. G., Balasubramanian, S., Boxer, S. G. 1989; 207 (1): 289-299

    Abstract

    Site-specific mutants of human myoglobin have been prepared in which lysine 45 is replaced by arginine (K45R) and aspartate 60 by glutamate (D60E), in order to examine the influence of these residues and their interaction on the dynamics of the protein. These proteins were studied by a variety of methods, including one and two-dimensional proton nuclear magnetic resonance spectroscopy, exchange kinetics for the distal and proximal histidine NH protons as a function of pH in the met cyano forms, flash photolysis of the CO forms, and ligand replacement kinetics. The electronic absorption and proton nuclear magnetic resonance spectra of the CO forms of these proteins are virtually identical, indicating that the structure of the heme pocket is unaltered by these mutations. There are, however, substantial changes in the dynamics of both CO binding and proton exchange for the mutant K45R, whereas the mutant D60E exhibits behavior indistinguishable from the reference human myoglobin. K45R has a faster CO bimolecular recombination rate and slower CO off-rate relative to the reference. The kinetics for CO binding are independent of pH (6.5 to 10) as well as ionic strength (0 to 1 M-NaCl). The exchange rate for the distal histidine NH is substantially lower for K45R than the reference, whereas the proximal histidine NH exchange rate is unaltered. The exchange behavior of the human proteins is similar to that reported for a comparison of the exchange rates for myoglobins having lysine at position 45 with sperm whale myoglobin, which has arginine at this position. This indicates that the differences in exchange rates reflects largely the Lys----Arg substitution. The lack of a simple correlation for the CO kinetics with this substitution means that these are sensitive to other factors as well. Specific kinetic models, whereby substitution of arginine for lysine at position 45 can affect ligand binding dynamics, are outlined. These experiments demonstrate that a relatively conservative change of a surface residue can substantially perturb ligand and proton exchange dynamics in a manner that is not readily predicted from the static structures.

    View details for Web of Science ID A1989U587200020

    View details for PubMedID 2544737

  • ELECTROSTATIC INTERACTIONS IN WILD-TYPE AND MUTANT RECOMBINANT HUMAN MYOGLOBINS BIOCHEMISTRY Varadarajan, R., Lambright, D. G., Boxer, S. G. 1989; 28 (9): 3771-3781

    Abstract

    Residue Val68 in human myoglobin has been replaced by Asn, Asp, and Glu with site-directed mutagenesis. Purified proteins were characterized by isoelectric focusing and by absorption, CD, and NMR spectroscopy. These studies demonstrated that Mb is able to tolerate substitution of the buried hydrophobic residue Val68 by Asn, Asp, and Glu. In the metaquo derivatives of the Glu and Asp mutants, the negative charge at residue 68 is stabilized by a favorable Coulombic interaction with the heme iron. In the absence of this interaction, as in the metcyano and ferrous deoxy derivatives, the relatively nonpolar protein interior cannot stabilize an isolated buried negative charge, and the carboxylate is either protonated or stabilized via a salt bridge with the nearby distal histidine. Hence in the Asp and Glu mutant proteins, both reduction and cyanide binding are accompanied by proton uptake by the protein. The apoproteins were prepared and reconstituted with the chlorophyll derivative zinc pyrochlorophyllide a. Absorption and fluorescence spectra were quite similar for wild-type and all mutant proteins reconstituted with this derivative. These results do not support the point charge model for the red shifts observed in the spectra of chlorophylls associated with photosynthetic proteins. From the pH dependence of the absorption spectrum of zinc pyrochlorophyllide a in the Glu mutant, the apparent pKa of the buried glutamate residue was estimated to be 8.9. This increase of 4.4 pH units, over the value for Glu in aqueous solution, provides a measure of the polarity of the protein interior.

    View details for Web of Science ID A1989U467600022

    View details for PubMedID 2751994

  • MULTINUCLEAR NMR-STUDIES OF DNA HAIRPINS .1. STRUCTURE AND DYNAMICS OF D(CGCGTTGTTCGCG) BIOCHEMISTRY Williamson, J. R., Boxer, S. G. 1989; 28 (7): 2819-2831

    Abstract

    The solution structure of the hairpin formed by d(CGCGTTGTTCGCG) has been examined in detail by a wide variety of NMR techniques. The hairpin was characterized by proton NMR to obtain interproton distances and torsion angle information. An energy-minimized model was constructed that is consistent with these data. The hairpin consists of a B-DNA stem of four C-G base pairs and a loop region consisting of five unpaired bases. Three bases in the 5' of the loop are stacked over the 3' end of the stem, and the other two bases in the 3' of the loop are stacked over the 5' end of the stem. The phosphorus NMR spectrum revealed a phosphate in the stem region with an unusual conformation, and two phosphates, P9 and P10, were found to undergo intermediate exchange between conformations. The hairpin was also synthesized with a carbon-13 label in each of the thymidine C6 carbons, and relaxation measurements were performed to determine the extent of internal motions in the loop region. The loop bases are more flexible than the stem bases and exhibit subnanosecond motions with an amplitude corresponding to diffusion in a cone of approximately 30 degrees.

    View details for Web of Science ID A1989U031400012

    View details for PubMedID 2742814

  • MULTINUCLEAR NMR-STUDIES OF DNA HAIRPINS .2. SEQUENCE-DEPENDENT STRUCTURAL VARIATIONS BIOCHEMISTRY Williamson, J. R., Boxer, S. G. 1989; 28 (7): 2831-2836

    Abstract

    The solution conformation of three related DNA hairpins, each with five bases in the loop, is investigated by proton and phosphorus 2D NMR methods. The sequences of the three oligomers are d(CGCGTTGTTCGCG), d(CGCGTTTGTCGCG), and d(CTGCTCTTGTTGAGCAG). One pair of hairpins shares the same stem sequence but differs in the loop, and the appearance of an unusual phosphate torsion in the stem is found to depend on the sequence in the loop of the hairpin. The second pair of hairpins shares the same loop region but differs in the stem sequence in that the base pair which closes the loop is a C-G or G-C pair. The pattern of NOEs reveals that the stacking arrangement in the loop region depends on the base pair that closes the stem. These results suggest that hairpin loop conformation and dynamics are sensitive to small changes in the loop and adjacent stem sequences. These findings are discussed in relation to sequence-dependent thermodynamic changes that have been observed in RNA hairpins.

    View details for Web of Science ID A1989U031400013

    View details for PubMedID 2742815

  • EFFECTS OF BURIED IONIZABLE AMINO-ACIDS ON THE REDUCTION POTENTIAL OF RECOMBINANT MYOGLOBIN SCIENCE Varadarajan, R., Zewert, T. E., Gray, H. B., Boxer, S. G. 1989; 243 (4887): 69-72

    Abstract

    The temperature dependences of the reduction potentials (E degrees') of wild-type human myoglobin (Mb) and three site-directed mutants have been measured by the use of thin-layer spectroelectrochemistry. Residue Val68, which is in van der Waals contact with the heme in Mb, has been replaced by Glu, Asp, and Asn. The changes in E degrees' and the standard entropy (delta S degrees') and enthalpy (delta H degrees') of reduction in the mutant proteins were determined relative to values for wild type; the change in E degrees' at 25 degrees C was about -200 millivolts for the Glu and Asp mutants, and about -80 millivolts for the Asn mutant. At pH 7.0, reduction of Fe(III) to Fe(II) in the Glu and Asp mutants is accompanied by uptake of a proton by the protein. These studies demonstrate that Mb can tolerate substitution of a buried hydrophobic group by potentially charged and polar residues and that such amino acid replacements can lead to substantial changes in the redox thermodynamics of the protein.

    View details for Web of Science ID A1989R646900031

    View details for PubMedID 2563171

  • ENERGETICS OF INITIAL CHARGE SEPARATION IN BACTERIAL PHOTOSYNTHESIS - THE TRIPLET DECAY-RATE IN VERY HIGH MAGNETIC-FIELDS BIOCHIMICA ET BIOPHYSICA ACTA Goldstein, R. A., Takiff, L., Boxer, S. G. 1988; 934 (2): 253-263
  • PHOSPHORESCENCE FROM THE PRIMARY ELECTRON-DONOR IN RHODOBACTER-SPHAEROIDES AND RHODOPSEUDOMONAS-VIRIDIS REACTION CENTERS BIOCHIMICA ET BIOPHYSICA ACTA Takiff, L., Boxer, S. G. 1988; 932 (3): 325-334
  • SYNTHESIS OF A THYMIDINE PHOSPHORAMIDITE LABELED WITH C-13 AT C6 - RELAXATION STUDIES OF THE LOOP REGION IN A C-13 LABELED DNA HAIRPIN NUCLEIC ACIDS RESEARCH Williamson, J. R., Boxer, S. G. 1988; 16 (4): 1529-1540

    Abstract

    A thymidine phosphoramidite labelled at C6 with 13C has been synthesized, and incorporated into a synthetic oligonucleotide, d(CGCGT*T*GT*T*CGCG), which adopts a hairpin conformation. NMR relaxation measurements indicate that internal motion may be present in the loop region of the oligonucleotide. The relaxation behavior of a the C6 carbon in a model compound, N,N-1,3 dimethylthymine is examined in detail as a function of magnetic field strength to determine relative contributions of various mechanisms to the relaxation. The relaxation behaviour of the labelled carbons in the oligonucleotide is discussed in relation to these measurements.

    View details for Web of Science ID A1988M280300022

    View details for PubMedID 3347496

  • STARK-EFFECT SPECTROSCOPY OF RHODOBACTER-SPHAEROIDES AND RHODOPSEUDOMONAS-VIRIDIS REACTION CENTERS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lockhart, D. J., Boxer, S. G. 1988; 85 (1): 107-111

    Abstract

    The nature of the initially excited state of the primary electron donor or special pair has been investigated by Stark effect spectroscopy for reaction centers from the photosynthetic bacteria Rhodopseudomonas viridis and Rhodobacter sphaeroides at 77 K. The data provide values for the magnitude of the difference in permanent dipole moment between the ground and excited state, [unk]Deltamu[unk], and the angle [unk] between Deltamu and the transition dipole moment for the electronic transition. [unk]Deltamu[unk] and [unk] for the lowest-energy singlet electronic transition associated with the special pair primary electron donor were found to be very similar for the two species. [unk]Deltamu[unk] for this transition is substantially larger than for the Q(y) transitions of the monomeric pigments in the reaction center or for pure monomeric bacteriochlorophylls, for which Stark data are also reported. We conclude that the excited state of the special pair has substantial charge-transfer character, and we suggest that charge separation in bacterial photosynthesis is initiated immediately upon photoexcitation of the special pair. Data for Rhodobacter sphaeroides between 340 and 1340 nm are presented and discussed in the context of the detection of charge-transfer states by Stark effect spectroscopy.

    View details for Web of Science ID A1988L864600024

    View details for PubMedID 16578825

  • EFFECTS OF NUCLEAR-SPIN POLARIZATION ON REACTION DYNAMICS IN PHOTOSYNTHETIC BACTERIAL REACTION CENTERS BIOPHYSICAL JOURNAL Goldstein, R. A., Boxer, S. G. 1987; 51 (6): 937-946

    Abstract

    Singlet-triplet mixing in the initial radical-pair state, P[unk]I[unk], of photosynthetic bacterial reaction centers is due to the hyperfine mechanism at low magnetic fields and both the hyperfine and Deltag mechanisms at high magnetic fields (>1 kG). Since the hyperfine field felt by the electron spins in P[unk]I[unk] is dependent upon the nuclear spin state in each radical, the relative probabilities of charge recombination to the triplet state of the primary electron donor, (3)PI, or the ground state, PI, will depend on the nuclear spin configuration. As a result these recombination products will have non-equilibrium distributions of nuclear spin states (nuclear spin polarization). This polarization will persist until the (3)PI state decays. In addition, due to unequal nuclear spin relaxation rates in the diamagnetic PI and paramagnetic (3)PI states, net polarization of the nuclear spins can result, especially in experiments that involve recycling of the system through the radical-pair state. This net polarization can persist for very long times, especially at low temperatures. Nuclear spin polarization can have consequences on any subsequent process that involves re-formation of the radical-pair state.Numerical calculations of the nuclear polarization caused by both of these mechanics are presented, including the effect of such polarization on subsequent yields of (3)PI, (3)PI decay rates, the decay rate of the radical pair, and saturation behavior. The effect of this polarization under certain circumstances can be very dramatic and can explain previously noted discrepancies between experiments and theories that do not include nuclear spin polarization effects. Our analysis suggests new classes of experiments and indicates the need to reinterpret some past experimental results.

    View details for Web of Science ID A1987H482800009

    View details for PubMedID 19431700

  • FLUORESCENCE LIFETIMES WITH A SYNCHROTRON SOURCE METHODS IN ENZYMOLOGY Boxer, S. G., BUCKS, R. R. 1986; 130: 484-493

    View details for Web of Science ID A1986E409500020

    View details for PubMedID 3773746

  • CLONING, EXPRESSION IN ESCHERICHIA-COLI, AND RECONSTITUTION OF HUMAN MYOGLOBIN PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Varadarajan, R., Szabo, A., Boxer, S. G. 1985; 82 (17): 5681-5684

    Abstract

    A full-length cDNA clone for human myoglobin has been isolated from a human skeletal muscle cDNA library. The clone as isolated has a cDNA insert approximately one kilobase long and has 5' and 3' untranslated regions of approximately 80 and 530 base pairs, respectively. The sequence of the translated region corresponds exactly to that predicted for human myoglobin. The cDNA was expressed in high yield in Escherichia coli as a fusion protein consisting of the first 31 amino acids of the phage lambda cII gene, the tetrapeptide Ile-Glu-Gly-Arg, and the myoglobin sequence by following the approach of Nagai and Thogersen [Nagai, K. & Thogersen, M. C. (1984) Nature (London) 309, 810-812]. The fusion product was isolated, reconstituted with heme, cleaved with trypsin, and purified to generate a protein whose properties are indistinguishable from those for authentic human myoglobin. Myoglobin can be readily prepared on a gram scale by using these methods.

    View details for Web of Science ID A1985AQM9400023

    View details for PubMedID 3898068

  • EFFECT OF MAGNETIC-FIELDS ON THE TRIPLET-STATE LIFETIME IN PHOTOSYNTHETIC REACTION CENTERS - EVIDENCE FOR THERMAL REPOPULATION OF THE INITIAL RADICAL PAIR PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chidsey, C. E., Takiff, L., Goldstein, R. A., Boxer, S. G. 1985; 82 (20): 6850-6854

    Abstract

    The lifetime of the molecular triplet state formed by recombination of the radical ion pair in quinonedepleted bacterial photosynthetic reaction centers is found to depend on applied magnetic field strength. It is suggested that this magnetic field effect results from thermally activated repopulation of the same radical ion pair that generates the triplet. Consistent with this hypothesis, the magnetic field effect on the triplet lifetime disappears at low temperature where the triplet state decays exclusively by ordinary intersystem crossing. This activated pathway for the decay of the triplet state can explain the strong temperature dependence of the triplet decay rate. A detailed theoretical treatment of the problem within a set of physically reasonable assumptions relates the observed temperature dependence of the triplet decay rate to the energy gap between the radical ion pair intermediate and the triplet state. This energy gap is estimated to be about 950 cm(-1) (0.12 eV). Combined with an estimate of the energy of the donor excited state, we obtain an energy gap between the excited singlet state of the donor and the radical ion pair of 2,250 cm(-1) (0.28 eV).

    View details for Web of Science ID A1985ATC6000028

    View details for PubMedID 16593615

  • MAGNETIC-FIELD DEPENDENCE OF RADICAL-PAIR DECAY KINETICS AND MOLECULAR TRIPLET QUANTUM YIELD IN QUINONE-DEPLETED REACTION CENTERS BIOCHIMICA ET BIOPHYSICA ACTA Chidsey, C. E., Kirmaier, C., HOLTEN, D., Boxer, S. G. 1984; 766 (2): 424-437
  • CHEMICALLY-INDUCED DYNAMIC NUCLEAR-POLARIZATION STUDIES OF YEAST TRANSFER RNAPHE BIOCHEMISTRY McCord, E. F., MORDEN, K. M., Tinoco, I., Boxer, S. G. 1984; 23 (9): 1935-1939

    Abstract

    Chemically induced dynamic nuclear polarization (CIDNP) has been observed from yeast tRNAPhe following reaction with photoexcited riboflavin. At 20 degrees C, several resonances of tRNA in the native form show polarization; previous work predicts that only guanosine and its derivatives in single-stranded regions are likely to become polarized [ McCord , E.F., Morden , K. M., Pardi , A., Tinoco , I., Jr., & Boxer, S. G. (1984) Biochemistry (preceding paper in this issue)]. The methyl protons of m22G -26 show strong negative spin polarization, indicating that this residue is accessible. The solvent accessibility of this residue has not been previously demonstrated. In addition, two positively polarized aromatic resonances are observed, which are likely due to two or more G(H8) protons, including those of G-20, m22G -26, and/or Gm-34. For temperatures below 50 degrees C, a negatively polarized signal in the aromatic region is shown to arise from cross relaxation with the methyl group protons of m22G -26. This indicates the proximity of an aromatic proton, probably H2 of A-44, to the methyl groups of m22G -26. At higher temperatures, the CIDNP spectra show polarization of several additional G resonances, including those of m2G -10. These changes in the CIDNP spectra reflect melting of the tertiary and secondary structure of the tRNA. This work is the first use of CIDNP to study a large nucleic acid molecule and exemplifies the value of this technique in probing single-stranded and solvent-accessible regions of tRNA.

    View details for Web of Science ID A1984SN53700007

    View details for PubMedID 6372863

  • EXCITATION TRANSPORT AND TRAPPING IN A SYNTHETIC CHLOROPHYLLIDE SUBSTITUTED HEMOGLOBIN - ORIENTATION OF THE CHLOROPHYLL-S1 TRANSITION DIPOLE BIOCHEMISTRY Moog, R. S., Kuki, A., Fayer, M. D., Boxer, S. G. 1984; 23 (7): 1564-1571

    Abstract

    Excitation transport in synthetic zinc chlorophyllide substituted hemoglobin has been observed by pico -second time-resolved fluorescence depolarization experiments. In this hybrid molecular system, two zinc chlorophyllide molecules are substituted into the beta-chains of hemoglobin, while deoxy hemes remain in the alpha-chains. The rate of excitation transfer between the two chlorophyllides is analyzed in terms of the distance and orientation dependences predicted by the F orster dipole-dipole theory. In this analysis, the beta-beta interchromophore geometry is assumed to be that of the deoxyhemoglobin crystal structure. When combined with steady-state fluorescence depolarization data of the complementary hybrid containing zinc chlorophyllide in the alpha-chains, these experiments provide the necessary information to determine the orientation of the S1 transition dipole moment in the zinc chlorophyllide molecule. We also find that the fluorescence lifetime of the zinc chlorophyllide is 1.42 ns when the heme is in the deoxy state but 3.75 ns when the heme is ligated to carbon monoxide. This is explained by irreversible excitation transfer from the S1 state of the zinc chlorophyllide to the lower energy excited states present in deoxy heme.

    View details for Web of Science ID A1984SK48400034

    View details for PubMedID 6722108

  • MAGNETIC-FIELD EFFECTS ON REACTION YIELDS IN THE SOLID-STATE - AN EXAMPLE FROM PHOTOSYNTHETIC REACTION CENTERS ANNUAL REVIEW OF PHYSICAL CHEMISTRY Boxer, S. G., Chidsey, C. E., Roelofs, M. G. 1983; 34: 389-417
  • CHLOROPHYLLIDE-SUBSTITUTED HEMOGLOBIN TETRAMERS AND HYBRIDS - PREPARATION, CHARACTERIZATION, AND ENERGY-TRANSFER BIOCHEMISTRY Kuki, A., Boxer, S. G. 1983; 22 (12): 2923-2933

    Abstract

    Three chlorophyllide-substituted human hemoglobin (Hb) complexes have been prepared: the tetrameric complex in which zinc pyrochlorophyllide alpha (ZnPChl alpha) is substituted for all four hemes and the two complementary hybrids in which ZnPChl alpha is substituted for heme in either the alpha- or beta-chains, while heme remains in the other chains. In each of these complexes, intramolecular Chl-Chl singlet energy transfer occurs. A variety of probes demonstrate that ZnPChl alpha-deoxyheme hybrids and the ZnPChl alpha-Hb complexes consistently exhibit properties associated with the well-known T-state tertiary and quaternary structure of deoxyHb itself. Using the known crystal structure of human deoxyHb, we have analyzed the steady-state fluorescence anisotropy of these complexes within the framework of the Förster energy-transfer theory. The result is the determination of the orientation of the Qy transition dipole moment of ZnPChl alpha. Nuclear magnetic resonance data for the hybrids offer insight into specific tertiary structural changes in the heme pocket surrounding the diamagnetic ZnPChl alpha, which accompany changes in the ligation state of the heme on the opposite chain.

    View details for Web of Science ID A1983QU52400022

    View details for PubMedID 6871173

  • USE OF LARGE MAGNETIC-FIELDS TO PROBE PHOTOINDUCED ELECTRON-TRANSFER REACTIONS - AN EXAMPLE FROM PHOTOSYNTHETIC REACTION CENTERS JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Boxer, S. G., Chidsey, C. E., Roelofs, M. G. 1982; 104 (5): 1452-1454
  • ORIENTED PROPERTIES OF THE CHLOROPHYLLS - ELECTRONIC ABSORPTION-SPECTROSCOPY OF ORTHORHOMBIC PYROCHLOROPHYLLIDE ALPHA-APOMYOGLOBIN SINGLE-CRYSTALS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES Boxer, S. G., Kuki, A., Wright, K. A., Katz, B. A., Xuong, N. H. 1982; 79 (4): 1121-1125

    Abstract

    The orientations of the transition dipole moments in chlorophyll (Chl) are among the most useful spectroscopic properties for determining macromolecular architecture in photosynthetic complexes; however, the relationships between these orientations and the Chl molecular geometry are unknown. In order to solve this problem, we have prepared single crystals of the synthetic 1:1 complex between pyrochlorophyllide a and apomyoglobin. The protein crystallizes readily in the orthorhombic (B) form, space group P2(1)2(1)2(1), and the unit cell dimensions are determined to be within 0.5% of those for native MetMb crystals of the same type. These green crystals are highly dichroic, and the strong absorption along the crystallographic a axis in the Q(y) band is red-shifted by about 9 nm, relative to the corresponding feature in a solution of the protein. Although the crystal structure for native Mb in this space group has not been determined, the direction cosines of the heme normal relative to the crystal axes have been measured. By using these values, an appropriate trigonometric analysis, and the measured polarized single-crystal spectra, the orientation of the Chl transition dipole moment for the Q(y) transition can be specified relative to the crystal axes. With the completion of the protein crystal structure, this result will lead directly to the orientations of the optical transition dipole moments relative to the molecular geometry. The effects of vibronic coupling and the protein environment on the absorption properties of Chl are discussed in detail.

    View details for Web of Science ID A1982NE35800036

    View details for PubMedID 16593158

  • ANISOTROPIC MAGNETIC-INTERACTIONS IN THE PRIMARY RADICAL ION-PAIR OF PHOTOSYNTHETIC REACTION CENTERS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES Boxer, S. G., Chidsey, C. E., Roelofs, M. G. 1982; 79 (15): 4632-4636

    Abstract

    The quantum yield of triplets formed by ion-pair recombination in quinone-depleted photosynthetic reaction centers is found to depend on their orientation in a magnetic field. This new effect is expected to be a general property of radical pair reactions in the solid state. For 0 < H < 1,000 G, the quantum yield anisotropy is caused by anisotropic electron dipole-electron dipole or nuclear hyperfine interactions, or both. For high fields it is dominated by the anisotropy of the difference g-tensor in the radical ion-pair. The magnitude and sign of the contribution of each interaction depend not only on the values of the principal components of each anisotropic tensor but also on the geometric relationship of the principal axes of each tensor to the transition dipole moment used to detect the yield. A detailed formalism is presented relating these quantities to the observed yield anisotropy. The expected magnitude of each anisotropic parameter is discussed. It is demonstrated that the field dependence of the yield anisotropy is consistent with these values for certain reaction center geometries.

    View details for Web of Science ID A1982PB77200023

    View details for PubMedID 16578764

  • CONTRIBUTIONS OF SPIN SPIN INTERACTIONS TO THE MAGNETIC-FIELD DEPENDENCE OF THE TRIPLET QUANTUM YIELD IN PHOTOSYNTHETIC REACTION CENTERS CHEMICAL PHYSICS LETTERS Roelofs, M. G., Chidsey, C. E., Boxer, S. G. 1982; 87 (6): 582-588
  • DEPENDENCE OF THE YIELD OF A RADICAL-PAIR REACTION IN THE SOLID-STATE ON ORIENTATION IN A MAGNETIC-FIELD JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Boxer, S. G., Chidsey, C. E., Roelofs, M. G. 1982; 104 (9): 2674-2675
  • CHLOROPHYLL AMINO-ACID INTERACTIONS IN SYNTHETIC MODELS ISRAEL JOURNAL OF CHEMISTRY Boxer, S. G., BUCKS, R. R. 1981; 21 (4): 259-264
  • UV-EXCIMER LASER CHEMICALLY-INDUCED DYNAMIC NUCLEAR-POLARIZATION OF AMINO-ACIDS AT 360-MHZ BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS McCord, E. F., Boxer, S. G. 1981; 100 (4): 1436-1443

    View details for Web of Science ID A1981LX31000003

    View details for PubMedID 7295309

  • SOLUTION PROPERTIES OF SYNTHETIC CHLOROPHYLLIDE-APOMYOGLOBIN AND "BACTERIOCHLOROPHYLLIDE-APOMYOGLOBIN COMPLEXES BIOCHEMISTRY Wright, K. A., Boxer, S. G. 1981; 20 (26): 7546-7556

    Abstract

    Well-defined 1:1 complexes have been formed between apomyoglobin (apoMb) and a number of chlorophyllide derivatives. The chlorophyllides substitute for heme in the pocket of myoglobin. These include magnesium chlorophyllide a, magnesium and zinc pyrochlorophyllide a, zinc pyrochlorophyllide b, zinc pyrochlorophyllide d, zinc pyromesochlorophyllide a, zinc 2-acetyl-2-devinylpyrochlorophyllide a, zinc protopyrochlorophyllide a, and zinc bacteriopyrochlorophyllide a. The effects of the protein on the electronic absorption, circular dichroism (CD), magnetic circular dichroism, and triplet state electron spin resonance spectra and fluorescence lifetimes in solution are compared with appropriate models in organic solvents. With the exception of the CD spectra, the protein causes shifts and intensity changes which are within the range observed for solvent effects. The CD spectra change substantially: the signs of several transitions are entirely reversed in the chlorins, and 3-6-fold intensity increases are observed with zinc bacteriochlorophyllide a. High-field 1H NMR spectra of ring current shifted Val-E11 methyl protons for the series porphyrin-, chlorin-, and bacteriochlorin-apoMb are used to establish the probable absolute orientation of the chromophore in the heme pocket. Doubled peaks in the NMR spectra of certain complexes are shown to arise from interconvertible species. The temperature dependence of the peak intensities and saturation transfer studies show that the species giving rise to the doubled peaks exchange on the time scale of about 1-60 s. Arguments are presented against inversion of the macrocycle in the heme pocket by either an inter- or an intramolecular mechanism as the origin of doubled peaks, and simple two-site exchange is ruled out by the NMR data. We suggest that the data are consistent with the idea that at least two slowly interconverting conformational substrates of the protein are populated, depending sensitively on small changes in rings I and II of the macrocycle and temperature.

    View details for Web of Science ID A1981MV24500033

    View details for PubMedID 7326244

  • LASER CHEMICALLY-INDUCED DYNAMIC NUCLEAR-POLARIZATION STUDY OF THE REACTION BETWEEN PHOTO-EXCITED FLAVINS AND TRYPTOPHAN DERIVATIVES AT 360 MHZ BIOCHEMISTRY McCord, E. F., BUCKS, R. R., Boxer, S. G. 1981; 20 (10): 2880-2888

    Abstract

    Chemically induced dynamic nuclear polarization (CIDNP) is generated when tryptophan (Trp), its derivatives, or Trp-containing peptides react with photoexcited flavins in a 360-MHz NMR spectrometer. In contrast to tyrosine (Tyr), we find that the nuclear polarization of Trp originates in an electron-transfer reaction. By use of a series of Trp derivatives, the unpaired spin-density distribution of the Trp radical cation and the ground-state NMR spectrum of Trp are analyzed in detail. The signs and the relative magnitudes of the proton isotropic hyperfine coupling constants for each position around the indole ring in the radical cation deduced from these measurements are the following: position 3 greater than 2 approximately 4 approximately 6 greater than 1 greater than 5 greater than 7, with positions 1, 2, 3, 4, and 6 positive, 5 negative, and 7 essentially zero. This result is inconsistent with most available calculations of the unpaired spin-density distribution but is compatible with the pattern of electrophilic aromatic substitution. The origin of this discrepancy is discussed in detail. Possible mechanistic complications in the reaction leading to CIDNP are discussed. The laser CIDNP spectra of the Trp-rich peptides gramicidins A and B are presented as examples of the resolution enhancement obtained with this technique.

    View details for Web of Science ID A1981LQ46200026

    View details for PubMedID 7248254

  • THE EFFECT OF LARGE MAGNETIC-FIELDS AND THE G-FACTOR DIFFERENCE ON THE TRIPLET POPULATION IN PHOTOSYNTHETIC REACTION CENTERS CHEMICAL PHYSICS LETTERS Chidsey, C. E., Roelofs, M. G., Boxer, S. G. 1980; 74 (1): 113-118
  • CHROMOPHORE ORGANIZATION IN PHOTOSYNTHETIC REACTION CENTERS - HIGH-RESOLUTION MAGNETOPHOTOSELECTION PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Boxer, S. G., Roelofs, M. G. 1979; 76 (11): 5636-5640

    Abstract

    The electron spin resonance spectrum of the triplet excited state of Rhodopseudomonas spheroides R-26 reaction centers has been studied after excitation with the polarized narrow-bandwidth output of a tunable dye laser from 520 to 670 nm. A theory is developed relating experimental observables to the angles between the electronic transition dipole moment of the excited chromophore and the principle magnetic axis system of the triplet state of the dimeric trap. Data is presented which demonstrates that the treatment is correct and useful, and angles are obtained for the Q(x) transitions of bacteriopheophytin. High-resolution magnetophotoselection data in the region of the bacteriochlorophyll Q(x) transitions can be combined with polarized photobleaching experiments to provide direct information on the structure of dimeric trap.

    View details for Web of Science ID A1979HW11500048

    View details for PubMedID 16592720

  • PHOTOPROCESSES IN COVALENTLY LINKED PYROCHLOROPHYLLIDE DIMER - TRIPLET-STATE FORMATION AND OPENING AND CLOSING OF HYDROXYLIC LINKAGES PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Periasamy, N., Linschitz, H., CLOSS, G. L., Boxer, S. G. 1978; 75 (6): 2563-2566

    Abstract

    Covalently linked pyrochlorophyllide a dimers exist in solution in either "pinned" or unfolded form, depending on the concentration of hydroxylic (H bonding) or non-H-bonding Lewis bases. Laser flash photolysis studies have been made on these dimer forms and on pyrochlorophyllide monomer at 30-nsec resolution. Spectra and recovery kinetics, including observation of both the triplet decay and ground state folding reactions, show that the flash transient obtained from the pinned form consists of a triplet and ground state moiety in the unpinned configuration. A charge-transfer excited state is not seen.

    View details for Web of Science ID A1978FF05900006

    View details for PubMedID 16592533

Conference Proceedings


  • Lipid-anchored DNA mediates vesicle fusion as observed by lipid and content mixing Chan, Y. M., van Lengerich, B., Boxer, S. G. SPRINGER. 2008: FA17-FA21

    Abstract

    A general method for synthesizing 5(')- and 3(')-coupled DNA-lipid conjugates has been developed and employed in DNA-mediated vesicle fusion. Vesicles presenting complementary DNA fuse, resulting in both outer and inner leaflet mixing as well as content mixing. Fusion is maximized using 5(')- and 3(')-coupled DNA on opposite vesicle partners, rather than only 5(')-coupled DNA, showing the importance of DNA orientation to the process. Lipid and content mixing assays show a dependence of fusion kinetics on the sequence and average number of DNA per vesicle. Vesicles without DNA or presenting noncomplementary sequences also appear to undergo some degree of lipid mixing or exchange, but no content mixing. Total lipid mixing appears to occur more efficiently than inner leaflet mixing and content mixing, and this may be explained by the observed nonspecific lipid mixing and/or the rise of a hemifused intermediate. The ability to control DNA sequence and the relative experimental simplicity of this system make it highly attractive to probe fundamental questions of membrane fusion using both ensemble and single vesicle assays.

    View details for DOI 10.1116/1.2889062

    View details for Web of Science ID 000264979100004

    View details for PubMedID 20408664

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