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Study of changes in conformation of proteins and RNA using x-ray scattering

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  • Caulobacter chromosome in vivo configuration matches model predictions for a supercoiled polymer in a cell-like confinement PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hong, S., Toro, E., Mortensen, K. I., de la Rosa, M. A., Doniach, S., Shapiro, L., Spakowitz, A. J., McAdams, H. H. 2013; 110 (5): 1674-1679

    Abstract

    We measured the distance between fluorescent-labeled DNA loci of various interloci contour lengths in Caulobacter crescentus swarmer cells to determine the in vivo configuration of the chromosome. For DNA segments less than about 300 kb, the mean interloci distances, , scale as n(0.22), where n is the contour length, and cell-to-cell distribution of the interloci distance r is a universal function of r/n(0.22) with broad cell-to-cell variability. For DNA segments greater than about 300 kb, the mean interloci distances scale as n, in agreement with previous observations. The 0.22 value of the scaling exponent for short DNA segments is consistent with theoretical predictions for a branched DNA polymer structure. Predictions from Brownian dynamics simulations of the packing of supercoiled DNA polymers in an elongated cell-like confinement are also consistent with a branched DNA structure, and simulated interloci distance distributions predict that confinement leads to "freezing" of the supercoiled configuration. Lateral positions of labeled loci at comparable positions along the length of the cell are strongly correlated when the longitudinal locus positions differ by <0.16 ?m. We conclude that the chromosome structure is supercoiled locally and elongated at large length scales and that substantial cell-to-cell variability in the interloci distances indicates that in vivo crowding prevents the chromosome from reaching an equilibrium arrangement. We suggest that the force causing rapid transport of loci remote from the parS centromere to the distal cell pole may arise from the release at the polar region of potential energy within the supercoiled DNA.

    View details for DOI 10.1073/pnas.1220824110

    View details for Web of Science ID 000314558100027

    View details for PubMedID 23319648

  • Salt dependence of the radius of gyration and flexibility of single-stranded DNA in solution probed by small-angle x-ray scattering PHYSICAL REVIEW E Sim, A. Y., Lipfert, J., Herschlag, D., Doniach, S. 2012; 86 (2)

    Abstract

    Short single-stranded nucleic acids are ubiquitous in biological processes; understanding their physical properties provides insights to nucleic acid folding and dynamics. We used small-angle x-ray scattering to study 8-100 residue homopolymeric single-stranded DNAs in solution, without external forces or labeling probes. Poly-T's structural ensemble changes with increasing ionic strength in a manner consistent with a polyelectrolyte persistence length theory that accounts for molecular flexibility. For any number of residues, poly-A is consistently more elongated than poly-T, likely due to the tendency of A residues to form stronger base-stacking interactions than T residues.

    View details for DOI 10.1103/PhysRevE.86.021901

    View details for Web of Science ID 000307277300008

    View details for PubMedID 23005779

  • Electrostatics of Nucleic Acid Folding under Conformational Constraint JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Anthony, P. C., Sim, A. Y., Chu, V. B., Doniach, S., Block, S. M., Herschlag, D. 2012; 134 (10): 4607-4614

    Abstract

    RNA folding is enabled by interactions between the nucleic acid and its ion atmosphere, the mobile sheath of aqueous ions that surrounds and stabilizes it. Understanding the ion atmosphere requires the interplay of experiment and theory. However, even an apparently simple experiment to probe the ion atmosphere, measuring the dependence of DNA duplex stability upon ion concentration and identity, suffers from substantial complexity, because the unfolded ensemble contains many conformational states that are difficult to treat accurately with theory. To minimize this limitation, we measured the unfolding equilibrium of a DNA hairpin using a single-molecule optical trapping assay, in which the unfolded state is constrained to a limited set of elongated conformations. The unfolding free energy increased linearly with the logarithm of monovalent cation concentration for several cations, such that smaller cations tended to favor the folded state. Mg(2+) stabilized the hairpin much more effectively at low concentrations than did any of the monovalent cations. Poisson-Boltzmann theory captured trends in hairpin stability measured for the monovalent cation titrations with reasonable accuracy, but failed to do so for the Mg(2+) titrations. This finding is consistent with previous work, suggesting that Poisson-Boltzmann and other mean-field theories fail for higher valency cations where ion-ion correlation effects may become significant. The high-resolution data herein, because of the straightforward nature of both the folded and the unfolded states, should serve as benchmarks for the development of more accurate electrostatic theories that will be needed for a more quantitative and predictive understanding of nucleic acid folding.

    View details for DOI 10.1021/ja208466h

    View details for Web of Science ID 000301990600036

    View details for PubMedID 22369617

  • AquaSAXS: a web server for computation and fitting of SAXS profiles with non-uniformally hydrated atomic models NUCLEIC ACIDS RESEARCH Poitevin, F., Orland, H., Doniach, S., Koehl, P., Delarue, M. 2011; 39: W184-W189

    Abstract

    Small Angle X-ray Scattering (SAXS) techniques are becoming more and more useful for structural biologists and biochemists, thanks to better access to dedicated synchrotron beamlines, better detectors and the relative easiness of sample preparation. The ability to compute the theoretical SAXS profile of a given structural model, and to compare this profile with the measured scattering intensity, yields crucial structural informations about the macromolecule under study and/or its complexes in solution. An important contribution to the profile, besides the macromolecule itself and its solvent-excluded volume, is the excess density due to the hydration layer. AquaSAXS takes advantage of recently developed methods, such as AquaSol, that give the equilibrium solvent density map around macromolecules, to compute an accurate SAXS/WAXS profile of a given structure and to compare it to the experimental one. Here, we describe the interface architecture and capabilities of the AquaSAXS web server (http://lorentz.dynstr.pasteur.fr/aquasaxs.php).

    View details for DOI 10.1093/nar/gkr430

    View details for Web of Science ID 000292325300031

    View details for PubMedID 21665925

  • Dissecting electrostatic screening, specific ion binding, and ligand binding in an energetic model for glycine riboswitch folding RNA-A PUBLICATION OF THE RNA SOCIETY Lipfert, J., Sim, A. Y., Herschlag, D., Doniach, S. 2010; 16 (4): 708-719

    Abstract

    Riboswitches are gene-regulating RNAs that are usually found in the 5'-untranslated regions of messenger RNA. As the sugar-phosphate backbone of RNA is highly negatively charged, the folding and ligand-binding interactions of riboswitches are strongly dependent on the presence of cations. Using small angle X-ray scattering (SAXS) and hydroxyl radical footprinting, we examined the cation dependence of the different folding stages of the glycine-binding riboswitch from Vibrio cholerae. We found that the partial folding of the tandem aptamer of this riboswitch in the absence of glycine is supported by all tested mono- and divalent ions, suggesting that this transition is mediated by nonspecific electrostatic screening. Poisson-Boltzmann calculations using SAXS-derived low-resolution structural models allowed us to perform an energetic dissection of this process. The results showed that a model with a constant favorable contribution to folding that is opposed by an unfavorable electrostatic term that varies with ion concentration and valency provides a reasonable quantitative description of the observed folding behavior. Glycine binding, on the other hand, requires specific divalent ions binding based on the observation that Mg(2+), Ca(2+), and Mn(2+) facilitated glycine binding, whereas other divalent cations did not. The results provide a case study of how ion-dependent electrostatic relaxation, specific ion binding, and ligand binding can be coupled to shape the energetic landscape of a riboswitch and can begin to be quantitatively dissected.

    View details for DOI 10.1261/rna.1985110

    View details for Web of Science ID 000275951000006

    View details for PubMedID 20194520

  • The Ligand-Free State of the TPP Riboswitch: A Partially Folded RNA Structure JOURNAL OF MOLECULAR BIOLOGY Ali, M., Lipfert, J., Seifert, S., Herschlag, D., Doniach, S. 2010; 396 (1): 153-165

    Abstract

    Riboswitches are elements of mRNA that regulate gene expression by undergoing structural changes upon binding of small ligands. Although the structures of several riboswitches have been solved with their ligands bound, the ligand-free states of only a few riboswitches have been characterized. The ligand-free state is as important for the functionality of the riboswitch as the ligand-bound form, but the ligand-free state is often a partially folded structure of the RNA, with conformational heterogeneity that makes it particularly challenging to study. Here, we present models of the ligand-free state of a thiamine pyrophosphate riboswitch that are derived from a combination of complementary experimental and computational modeling approaches. We obtain a global picture of the molecule using small-angle X-ray scattering data and use an RNA structure modeling software, MC-Sym, to fit local structural details to these data on an atomic scale. We have used two different approaches to obtaining these models. Our first approach develops a model of the RNA from the structures of its constituent junction fragments in isolation. The second approach treats the RNA as a single entity, without bias from the structure of its individual constituents. We find that both approaches give similar models for the ligand-free form, but the ligand-bound models differ for the two approaches, and only the models from the second approach agree with the ligand-bound structure known previously from X-ray crystallography. Our models provide a picture of the conformational changes that may occur in the riboswitch upon binding of its ligand. Our results also demonstrate the power of combining experimental small-angle X-ray scattering data with theoretical structure prediction tools in the determination of RNA structures beyond riboswitches.

    View details for DOI 10.1016/j.jmb.2009.11.030

    View details for Web of Science ID 000274766500013

    View details for PubMedID 19925806

  • Combining Single-Molecule Optical Trapping and Small-Angle X-Ray Scattering Measurements to Compute the Persistence Length of a Protein ER/K alpha-Helix BIOPHYSICAL JOURNAL Sivaramakrishnan, S., Sung, J., Ali, M., Doniach, S., Flyvbjerg, H., Spudich, J. A. 2009; 97 (11): 2993-2999

    Abstract

    A relatively unknown protein structure motif forms stable isolated single alpha-helices, termed ER/K alpha-helices, in a wide variety of proteins and has been shown to be essential for the function of some molecular motors. The flexibility of the ER/K alpha-helix determines whether it behaves as a force transducer, rigid spacer, or flexible linker in proteins. In this study, we quantify this flexibility in terms of persistence length, namely the length scale over which it is rigid. We use single-molecule optical trapping and small-angle x-ray scattering, combined with Monte Carlo simulations to demonstrate that the Kelch ER/K alpha-helix behaves as a wormlike chain with a persistence length of approximately 15 nm or approximately 28 turns of alpha-helix. The ER/K alpha-helix length in proteins varies from 3 to 60 nm, with a median length of approximately 5 nm. Knowledge of its persistence length enables us to define its function as a rigid spacer in a translation initiation factor, as a force transducer in the mechanoenzyme myosin VI, and as a flexible spacer in the Kelch-motif-containing protein.

    View details for DOI 10.1016/j.bpj.2009.09.009

    View details for Web of Science ID 000272274500017

    View details for PubMedID 19948129

  • Do conformational biases of simple helical junctions influence RNA folding stability and specificity? RNA-A PUBLICATION OF THE RNA SOCIETY Chu, V. B., Lipfert, J., Bai, Y., Pande, V. S., Doniach, S., Herschlag, D. 2009; 15 (12): 2195-2205

    Abstract

    Structured RNAs must fold into their native structures and discriminate against a large number of alternative ones, an especially difficult task given the limited information content of RNA's nucleotide alphabet. The simplest motifs within structured RNAs are two helices joined by nonhelical junctions. To uncover the fundamental behavior of these motifs and to elucidate the underlying physical forces and challenges faced by structured RNAs, we computationally and experimentally studied a tethered duplex model system composed of two helices joined by flexible single- or double-stranded polyethylene glycol tethers, whose lengths correspond to those typically observed in junctions from structured RNAs. To dissect the thermodynamic properties of these simple motifs, we computationally probed how junction topology, electrostatics, and tertiary contact location influenced folding stability. Small-angle X-ray scattering was used to assess our predictions. Single- or double-stranded junctions, independent of sequence, greatly reduce the space of allowed helical conformations and influencing the preferred location and orientation of their adjoining helices. A double-stranded junction guides the helices along a hinge-like pathway. In contrast, a single-stranded junction samples a broader set of conformations and has different preferences than the double-stranded junction. In turn, these preferences determine the stability and distinct specificities of tertiary structure formation. These sequence-independent effects suggest that properties as simple as a junction's topology can generally define the accessible conformational space, thereby stabilizing desired structures and assisting in discriminating against misfolded structures. Thus, junction topology provides a fundamental strategy for transcending the limitations imposed by the low information content of RNA primary sequence.

    View details for DOI 10.1261/rna.1747509

    View details for Web of Science ID 000272169000011

    View details for PubMedID 19850914

  • Mixing and Matching Detergents for Membrane Protein NMR Structure Determination JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Columbus, L., Lipfert, J., Jambunathan, K., Fox, D. A., Sim, A. Y., Doniach, S., Lesley, S. A. 2009; 131 (21): 7320-7326

    Abstract

    One major obstacle to membrane protein structure determination is the selection of a detergent micelle that mimics the native lipid bilayer. Currently, detergents are selected by exhaustive screening because the effects of protein-detergent interactions on protein structure are poorly understood. In this study, the structure and dynamics of an integral membrane protein in different detergents is investigated by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy and small-angle X-ray scattering (SAXS). The results suggest that matching of the micelle dimensions to the protein's hydrophobic surface avoids exchange processes that reduce the completeness of the NMR observations. Based on these dimensions, several mixed micelles were designed that improved the completeness of NMR observations. These findings provide a basis for the rational design of mixed micelles that may advance membrane protein structure determination by NMR.

    View details for DOI 10.1021/ja808776j

    View details for Web of Science ID 000266484900031

    View details for PubMedID 19425578

  • Riboswitch conformations revealed by small-angle X-ray scattering. Methods in molecular biology (Clifton, N.J.) Lipfert, J., Herschlag, D., Doniach, S. 2009; 540: 141-159

    Abstract

    Riboswitches are functional RNA molecules that control gene expression through conformational changes in response to small-molecule ligand binding. In addition, riboswitch 3D structure, like that of other RNA molecules, is dependent on cation-RNA interactions as the RNA backbone is highly negatively charged. Here, we show how small-angle X-ray scattering (SAXS) can be used to probe RNA conformations as a function of ligand and ion concentration. In a recent study of a glycine-binding tandem aptamer from Vibrio cholerae, we have used SAXS data and thermodynamic modeling to investigate how Mg(2+)-dependent folding and glycine binding are energetically coupled. In addition, we have employed ab initio shape reconstruction algorithms to obtain low-resolution models of the riboswitch structure from SAXS data under different solution conditions.

    View details for DOI 10.1007/978-1-59745-558-9_11

    View details for PubMedID 19381558

  • USE OF SMALL ANGLE X-RAY SCATTERING (SAXS) TO CHARACTERIZE CONFORMATIONAL STATES OF FUNCTIONAL RNAs METHODS IN ENZYMOLOGY, VOL 469: BIOPHYSICAL, CHEMICAL, AND FUNCTIONAL PROBES OF RNA STRUCTURE, INTERACTIONS AND FOLDING, PT B Doniach, S., Lipfert, J. 2009; 469: 237-251

    Abstract

    Small-angle X-ray scattering (SAXS) is emerging as an important technique to characterize the structure of RNA molecules. While lower in resolution than X-ray crystallography or NMR spectroscopy, SAXS has the great advantage to have virtually no molecular weight limitations and does not require crystallization. In addition, SAXS can be readily applied under a large range of solution conditions, allowing to monitor RNA folding, ligand binding, and to characterize partially folded intermediates. Here, we review how the development of SAXS as a structural technique is driven by advances in computer algorithms that allow to reconstruct low-resolution electron density maps ab initio from scattering profiles. In addition, we delineate how these low-resolution models can be used in free energy electrostatics calculations. Finally, we discuss how one can exploit the hierarchical nature of RNA folding by combining the low resolution, global information provided by SAXS with local information on RNA structure, from either experiments or state-of-the-art RNA structure prediction algorithms, to further increase the resolution and quality of models obtained from SAXS.

    View details for DOI 10.1016/S0076-6879(09)69011-X

    View details for Web of Science ID 000272797000011

    View details for PubMedID 20946792

  • A repulsive field: advances in the electrostatics of the ion atmosphere CURRENT OPINION IN CHEMICAL BIOLOGY Chu, V. B., Bai, Y., Lipfert, J., Herschlag, D., Doniach, S. 2008; 12 (6): 619-625

    Abstract

    The large electrostatic repulsion arising from the negatively charged backbone of RNA molecules presents a large barrier to folding. Solution counterions assist in the folding process by screening this electrostatic repulsion. While early research interpreted the effect of these counterions in terms of an empirical ligand-binding model, theories based on physical models have supplanted them and revised our view of the roles that ions play in folding. Instead of specific ion-binding sites, most ions in solution interact inside an 'ion atmosphere'--a fluctuating cloud of nonspecifically associated ions surrounding any charged molecule. Recent advances in experiments have begun the task of characterizing the ion atmosphere, yielding valuable data that have revealed deficiencies in Poisson-Boltzmann theory, the most widely used theory of the ion atmosphere. The continued development of experiments will help guide the development of improved theories, with the ultimate goal of understanding RNA folding and function and nucleic acid/protein interactions from a quantitative perspective.

    View details for DOI 10.1016/j.cbpa.2008.10.010

    View details for Web of Science ID 000262541300004

    View details for PubMedID 19081286

  • Critical assessment of nucleic acid electrostatics via experimental and computational investigation of an unfolded state ensemble JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Bai, Y., Chu, V. B., Lipfert, J., Pande, V. S., Herschlag, D., Doniach, S. 2008; 130 (37): 12334-12341

    Abstract

    Electrostatic forces, acting between helices and modulated by the presence of the ion atmosphere, are key determinants in the energetic balance that governs RNA folding. Previous studies have employed Poisson-Boltzmann (PB) theory to compute the energetic contribution of these forces in RNA folding. However, the complex interaction of these electrostatic forces with RNA features such as tertiary contact formation, specific ion-binding, and complex interhelical junctions present in prior studies precluded a rigorous evaluation of PB theory, especially in physiologically important Mg(2+) solutions. To critically assess PB theory, we developed a model system that isolates these electrostatic forces. The model system, composed of two DNA duplexes tethered by a polyethylene glycol junction, is an analog for the unfolded state of canonical helix-junction-helix motifs found in virtually all structured RNAs. This model system lacks the complicating features that have precluded a critical assessment of PB in prior studies, ensuring that interhelical electrostatic forces dominate the behavior of the system. The system's simplicity allows PB predictions to be directly compared with small-angle X-ray scattering experiments over a range of monovalent and divalent ion concentrations. These comparisons indicate that PB is a reasonable description of the underlying electrostatic energies for monovalent ions, but large deviations are observed for divalent ions. The validation of PB for monovalent solutions allows analysis of the change in the conformational ensemble of this simple motif as salt concentration is changed. Addition of ions allows the motif to sample more compact microstates, increasing its conformational entropy. The increase of conformational entropy presents an additional barrier to folding by stabilizing the unfolded state. Neglecting this effect will adversely impact the accuracy of folding analyses and models.

    View details for DOI 10.1021/ja800854u

    View details for Web of Science ID 000259139900046

    View details for PubMedID 18722445

  • The complete VS ribozyme in solution studied by small-angle X-ray scattering STRUCTURE Lipfert, J., Ouellet, J., Norman, D. G., Doniach, S., Lilley, D. M. 2008; 16 (9): 1357-1367

    Abstract

    We have used small-angle X-ray solution scattering to obtain ab initio shape reconstructions of the complete VS ribozyme. The ribozyme occupies an electron density envelope with an irregular shape, into which helical sections have been fitted. The ribozyme is built around a core comprising a near-coaxial stack of three helices, organized by two three-way helical junctions. An additional three-way junction formed by an auxiliary helix directs the substrate stem-loop, juxtaposing the cleavage site with an internal loop to create the active complex. This is consistent with the current view of the probable mechanism of trans-esterification in which adenine and guanine nucleobases contributed by the interacting loops combine in general acid-base catalysis.

    View details for DOI 10.1016/j.str.2008.07.007

    View details for Web of Science ID 000259164500011

    View details for PubMedID 18786398

  • Dynamic charge interactions create surprising rigidity in the ER/K alpha-helical protein motif PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sivaramakrishnan, S., Spink, B. J., Sim, A. Y., Doniach, S., Spudich, J. A. 2008; 105 (36): 13356-13361

    Abstract

    Protein alpha-helices are ubiquitous secondary structural elements, seldom considered to be stable without tertiary contacts. However, amino acid sequences in proteins that are based on alternating repeats of four glutamic acid (E) residues and four positively charged residues, a combination of arginine (R) and lysine (K), have been shown to form stable alpha-helices in a few proteins, in the absence of tertiary interactions. Here, we find that this ER/K motif is more prevalent than previously reported, being represented in proteins of diverse function from archaea to humans. By using molecular dynamics (MD) simulations, we characterize a dynamic pattern of side-chain interactions that extends along the backbone of ER/K alpha-helices. A simplified model predicts that side-chain interactions alone contribute substantial bending rigidity (0.5 pN/nm) to ER/K alpha-helices. Results of small-angle x-ray scattering (SAXS) and single-molecule optical-trap analyses are consistent with the high bending rigidity predicted by our model. Thus, the ER/K alpha-helix is an isolated secondary structural element that can efficiently span long distances in proteins, making it a promising tool in designing synthetic proteins. We propose that the significant rigidity of the ER/K alpha-helix can help regulate protein function, as a force transducer between protein subdomains.

    View details for DOI 10.1073/pnas.0806256105

    View details for Web of Science ID 000259251700034

    View details for PubMedID 18768817

  • Long single alpha-helical tail domains bridge the gap between structure and function of myosin VI NATURE STRUCTURAL & MOLECULAR BIOLOGY Spink, B. J., Sivaramakrishnan, S., Lipfert, J., Doniach, S., Spudich, J. A. 2008; 15 (6): 591-597

    Abstract

    Myosin VI has challenged the lever arm hypothesis of myosin movement because of its ability to take approximately 36-nm steps along actin with a canonical lever arm that seems to be too short to allow such large steps. Here we demonstrate that the large step of dimeric myosin VI is primarily made possible by a medial tail in each monomer that forms a rare single alpha-helix of approximately 10 nm, which is anchored to the calmodulin-bound IQ domain by a globular proximal tail. With the medial tail contributing to the approximately 36-nm step, rather than dimerizing as previously proposed, we show that the cargo binding domain is the dimerization interface. Furthermore, the cargo binding domain seems to be folded back in the presence of the catalytic head, constituting a potential regulatory mechanism that inhibits dimerization.

    View details for DOI 10.1038/nsmb.1429

    View details for Web of Science ID 000256388900013

    View details for PubMedID 18511944

  • Quantitative and comprehensive decomposition of the ion atmosphere around nucleic acids JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Bai, Y., Greenfeld, M., Travers, K. J., Chu, V. B., Lipfert, J., Doniach, S., Herschlag, D. 2007; 129 (48): 14981-14988

    Abstract

    The ion atmosphere around nucleic acids critically affects biological and physical processes such as chromosome packing, RNA folding, and molecular recognition. However, the dynamic nature of the ion atmosphere renders it difficult to characterize. The basic thermodynamic description of this atmosphere, a full accounting of the type and number of associated ions, has remained elusive. Here we provide the first complete accounting of the ion atmosphere, using buffer equilibration and atomic emission spectroscopy (BE-AES) to accurately quantitate the cation association and anion depletion. We have examined the influence of ion size and charge on ion occupancy around simple, well-defined DNA molecules. The relative affinity of monovalent and divalent cations correlates inversely with their size. Divalent cations associate preferentially over monovalent cations; e.g., with Na+ in 4-fold excess of Mg2+ (20 vs 5 mM), the ion atmosphere nevertheless has 3-fold more Mg2+ than Na+. Further, the dicationic polyamine putrescine2+ does not compete effectively for association relative to divalent metal ions, presumably because of its lower charge density. These and other BE-AES results can be used to evaluate and guide the improvement of electrostatic treatments. As a first step, we compare the BE-AES results to predictions from the widely used nonlinear Poisson Boltzmann (NLPB) theory and assess the applicability and precision of this theory. In the future, BE-AES in conjunction with improved theoretical models, can be applied to complex binding and folding equilibria of nucleic acids and their complexes, to parse the electrostatic contribution from the overall thermodynamics of important biological processes.

    View details for DOI 10.1021/ja075020g

    View details for Web of Science ID 000251293500034

    View details for PubMedID 17990882

  • Evaluation of ion binding to DNA duplexes using a size-modified Poisson-Boltzmann theory BIOPHYSICAL JOURNAL Chu, V. B., Bai, Y., Lipfert, J., Herschlag, D., Doniach, S. 2007; 93 (9): 3202-3209

    Abstract

    Poisson-Boltzmann (PB) theory is among the most widely applied electrostatic theories in biological and chemical science. Despite its reasonable success in explaining a wide variety of phenomena, it fails to incorporate two basic physical effects, ion size and ion-ion correlations, into its theoretical treatment. Recent experimental work has shown significant deviations from PB theory in competitive monovalent and divalent ion binding to a DNA duplex. The experimental data for monovalent binding are consistent with a hypothesis that attributes these deviations to counterion size. To model the observed differences, we have generalized an existing size-modified Poisson-Boltzmann (SMPB) theory and developed a new numerical implementation that solves the generalized theory around complex, atomistic representations of biological molecules. The results of our analysis show that good agreement to data at monovalent ion concentrations up to approximately 150 mM can be attained by adjusting the ion-size parameters in the new size-modified theory. SMPB calculations employing calibrated ion-size parameters predict experimental observations for other nucleic acid structures and salt conditions, demonstrating that the theory is predictive. We are, however, unable to model the observed deviations in the divalent competition data with a theory that only accounts for size but neglects ion-ion correlations, highlighting the need for theoretical descriptions that further incorporate ion-ion correlations. The accompanying numerical solver has been released publicly, providing the general scientific community the ability to compute SMPB solutions around a variety of different biological structures with only modest computational resources.

    View details for DOI 10.1529/biophysj.106.099168

    View details for Web of Science ID 000250199300024

    View details for PubMedID 17604318

  • Size and shape of detergent micelles determined by small-angle x-ray scattering JOURNAL OF PHYSICAL CHEMISTRY B Lipfert, J., Columbus, L., Chu, V. B., Lesley, S. A., Doniach, S. 2007; 111 (43): 12427-12438

    Abstract

    We present a systematic analysis of the aggregation number and shape of micelles formed by nine detergents commonly used in the study of membrane proteins. Small-angle X-ray scattering measurements are reported for glucosides with 8 and 9 alkyl carbons (OG/NG), maltosides and phosphocholines with 10 and 12 alkyl carbons (DM/DDM and FC-10/FC-12), 1,2-dihexanoyl-sn-glycero-phosphocholine (DHPC), 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LPPG), and 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS). The SAXS intensities are well described by two-component ellipsoid models, with a dense outer shell corresponding to the detergent head groups and a less electron dense hydrophobic core. These models provide an intermediate resolution view of micelle size and shape. In addition, we show that Guinier analysis of the forward scattering intensity can be used to obtain an independent and model-free measurement of the micelle aggregation number and radius of gyration. This approach has the advantage of being easily generalizable to protein-detergent complexes, where simple geometric models are inapplicable. Furthermore, we have discovered that the position of the second maximum in the scattering intensity provides a direct measurement of the characteristic head group-head group spacing across the micelle core. Our results for the micellar aggregation numbers and dimensions agree favorably with literature values as far as they are available. We de novo determine the shape of FC-10, FC-12, DM, LPPG, and CHAPS micelles and the aggregation numbers of FC-10 and OG to be ca. 50 and 250, respectively. Combined, these data provide a comprehensive view of the determinants of micelle formation and serve as a starting point to correlate detergent properties with detergent-protein interactions.

    View details for DOI 10.1021/jp073016l

    View details for Web of Science ID 000250556600013

    View details for PubMedID 17924686

  • MinActionPath: maximum likelihood trajectory for large-scale structural transitions in a coarse-grained locally harmonic energy landscape NUCLEIC ACIDS RESEARCH Franklin, J., Koehl, P., Doniach, S., Delarue, M. 2007; 35: W477-W482

    Abstract

    The non-linear problem of simulating the structural transition between two known forms of a macromolecule still remains a challenge in structural biology. The problem is usually addressed in an approximate way using 'morphing' techniques, which are linear interpolations of either the Cartesian or the internal coordinates between the initial and end states, followed by energy minimization. Here we describe a web tool that implements a new method to calculate the most probable trajectory that is exact for harmonic potentials; as an illustration of the method, the classical Calpha-based Elastic Network Model (ENM) is used both for the initial and the final states but other variants of the ENM are also possible. The Langevin equation under this potential is solved analytically using the Onsager and Machlup action minimization formalism on each side of the transition, thus replacing the original non-linear problem by a pair of linear differential equations joined by a non-linear boundary matching condition. The crossover between the two multidimensional energy curves around each state is found numerically using an iterative approach, producing the most probable trajectory and fully characterizing the transition state and its energy. Jobs calculating such trajectories can be submitted on-line at: http://lorentz.dynstr.pasteur.fr/joel/index.php.

    View details for DOI 10.1093/nar/gkm342

    View details for Web of Science ID 000255311500090

    View details for PubMedID 17545201

  • Structural transitions and thermodynamics of a glycine-dependent riboswitch from Vibrio cholerae JOURNAL OF MOLECULAR BIOLOGY Lipfert, J., Das, R., Chu, V. B., Kudaravalli, M., Boyd, N., Herschlag, D., Doniach, S. 2007; 365 (5): 1393-1406

    Abstract

    Riboswitches are complex folded RNA domains found in noncoding regions of mRNA that regulate gene expression upon small molecule binding. Recently, Breaker and coworkers reported a tandem aptamer riboswitch (VCI-II) that binds glycine cooperatively. Here, we use hydroxyl radical footprinting and small-angle X-ray scattering (SAXS) to study the conformations of this tandem aptamer as a function of Mg(2+) and glycine concentration. We fit a simple three-state thermodynamic model that describes the energetic coupling between magnesium-induced folding and glycine binding. Furthermore, we characterize the structural conformations of each of the three states: In low salt with no magnesium present, the VCI-II construct has an extended overall conformation, presumably representing unfolded structures. Addition of millimolar concentrations of Mg(2+) in the absence of glycine leads to a significant compaction and partial folding as judged by hydroxyl radical protections. In the presence of millimolar Mg(2+) concentrations, the tandem aptamer binds glycine cooperatively. The glycine binding transition involves a further compaction, additional tertiary packing interactions and further uptake of magnesium ions relative to the state in high Mg(2+) but no glycine. Employing density reconstruction algorithms, we obtain low resolution 3-D structures for all three states from the SAXS measurements. These data provide a first glimpse into the structural conformations of the VCI-II aptamer, establish rigorous constraints for further modeling, and provide a framework for future mechanistic studies.

    View details for DOI 10.1016/j.jmb.2006.10.022

    View details for Web of Science ID 000243749600013

    View details for PubMedID 17118400

  • Small-angle X-ray scattering from RNA, proteins, and protein complexes ANNUAL REVIEW OF BIOPHYSICS AND BIOMOLECULAR STRUCTURE Lipfert, J., Doniach, S. 2007; 36: 307-327

    Abstract

    Small-angle X-ray scattering (SAXS) is increasingly used to characterize the structure and interactions of biological macromolecules and their complexes in solution. Although still a low-resolution technique, the advent of high-flux synchrotron sources and the development of algorithms for the reconstruction of 3-D electron density maps from 1-D scattering profiles have made possible the generation of useful low-resolution molecular models from SAXS data. Furthermore, SAXS is well suited for the study of unfolded or partially folded conformational ensembles as a function of time or solution conditions. Here, we review recently developed algorithms for 3-D structure modeling and applications to protein complexes. Furthermore, we discuss the emerging use of SAXS as a tool to study membrane protein-detergent complexes. SAXS is proving useful to study the folding of functional RNA molecules, and finally we discuss uses of SAXS to study ensembles of denatured proteins.

    View details for DOI 10.1146/annurev.biophys.36.040306.132655

    View details for Web of Science ID 000247773000015

    View details for PubMedID 17284163

  • Dynamic bond constraints in protein Langevin dynamics JOURNAL OF CHEMICAL PHYSICS Franklin, J., Doniach, S. 2006; 124 (15)

    Abstract

    Bond constraint algorithms for molecular dynamics typically take, as the target constraint lengths, the values of the equilibrium bond lengths defined in the potential. In Langevin form, the equations of motion are temperature dependent, which gives the average value for the individual bond lengths a temperature dependence. In addition to this, locally constant force fields can shift the local equilibrium bond lengths. To restore the average bond lengths in constrained integration to their unconstrained values, we suggest changing the constraint length used by popular constraint methods such as RATTLE [H. C. Andersen, J. Comput. Phys. 52, 23 (1983)] at each step. This allows us to more accurately capture the equilibrium bond length changes (with respect to the potential) due to the local equilibration and temperature effects. In addition, the approximations to the unconstrained nonbonded energies are closer using the dynamic constraint method than a traditional fixed constraint algorithm. The mechanism for finding the new constrained lengths involves one extra calculation of the bonded components of the force, and therefore adds O(N) time to the constraint algorithm. Since most molecular dynamics calculations are dominated by the O(N2) nonbonded forces, this new method does not take significantly more time than a fixed constraint algorithm.

    View details for DOI 10.1063/1.2178325

    View details for Web of Science ID 000236969500049

    View details for PubMedID 16674259

  • Adaptive time stepping in biomolecular dynamics JOURNAL OF CHEMICAL PHYSICS Franklin, J., Doniach, S. 2005; 123 (12)

    Abstract

    We present an adaptive time stepping scheme based on the extrapolative method of Barth and Schlick [LN, J. Chem. Phys. 109, 1633 (1998)] to numerically integrate the Langevin equation with a molecular-dynamics potential. This approach allows us to use (on average) a time step for the strong nonbonded force integration corresponding to half the period of the fastest bond oscillation, without compromising the slow degrees of freedom in the problem. We show with simple examples how the dynamic step size stabilizes integration operators, and discuss some of the limitations of such stability. The method introduced uses a slightly more accurate inner integrator than LN to accommodate the larger steps. The adaptive time step approach reproduces temporal features of the bovine pancreatic trypsin inhibitor (BPTI) test system (similar to the one used in the original introduction of LN) compared to short-time integrators, but with energies that are shifted with respect to both LN, and traditional stochastic versions of Verlet. Although the introduction of longer steps has the effect of systematically heating the bonded components of the potential, the temporal fluctuations of the slow degrees of freedom are reproduced accurately. The purpose of this paper is to display a mechanism by which the resonance traditionally associated with using time steps corresponding to half the period of oscillations in molecular dynamics can be avoided. This has theoretical utility in terms of designing numerical integration schemes--the key point is that by factoring a propagator so that time steps are not constant one can recover stability with an overall (average) time step at a resonance frequency. There are, of course, limitations to this approach associated with the complicated, nonlinear nature of the molecular-dynamics (MD) potential (i.e., it is not as straightforward as the linear test problem we use to motivate the method). While the basic notion remains in the full Newtonian problem, it is easier to see the effects when damping is considered to be physical--that is, we do not view our method as a perturbation of Newtonian dynamics, we associate the damping with the environment, for example, a water bath (with gamma approximately 90 ps(-1)) [Zagrovic and Pande, J. Comp. Chem. 24, 1432 (2003)]. All stochastic approaches to MD are stabilized by large physical damping, but here, we are really using it only to show that the resonance frequency can be obtained. Another simplifying assumption used in this paper is "heavy" hydrogen (we take the hydrogen mass to be 10 amu)--the view here is that we are interested primarily in the slowest degrees of freedom, and this approach has effects similar to bond freezing and united atom treatments of hydrogen. So from the point of view of biomolecular applications, the method described here is best suited to studies in which water is not explicit (so that damping in the problem can really be viewed as environmental interaction), and the interest is in slow dynamics where the effects of hydrogen are neglectable. There are a number of parameters in the LN method and the one derived here, and we cannot in a short paper address all adjustments, so our primary goal as a first pass is to show that stability can be recovered for a set of numerically forced (and hence artificial) bond oscillations, and compare stability to fixed-step methods.

    View details for DOI 10.1063/1.1997137

    View details for Web of Science ID 000232206500067

    View details for PubMedID 16392529

  • Protein misfolding and amyloid formation for the peptide GNNQQNY from yeast prion protein Sup35: Simulation by reaction path annealing JOURNAL OF MOLECULAR BIOLOGY Lipfert, J., Franklin, J., Wu, F., Doniach, S. 2005; 349 (3): 648-658

    Abstract

    We study the early steps of amyloid formation of the seven residue peptide GNNQQNY from yeast prion-like protein Sup35 by simulating the random coil to beta-sheet and alpha-helix to beta-sheet transition both in the absence and presence of a cross-beta amyloid nucleus. The simulation method at atomic resolution employs a new implementation of a Langevin dynamics "reaction path annealing" algorithm. The results indicate that the presence of amyloid-like cross-beta-sheet strands both facilitates the transition into the cross-beta conformation and substantially lowers the free energy barrier for this transition. This model systems allows us to investigate the energetic and kinetic details of this transition, which is consistent with an auto-catalyzed, nucleation-like mechanism for the formation of beta-amyloid. In particular, we find that electrostatic interactions of peptide backbone dipoles contribute significantly to the stability of the beta-amyloid state. Furthermore, we find water exclusion and interactions of polar side-chains to be driving forces of amyloid formation: the cross-beta conformation is stabilized by burial of polar side-chains and inter-residue hydrogen bonds in the presence of an amyloid-like "seed". These findings are in support of a "dry, polar zipper model" of amyloid formation.

    View details for DOI 10.1016/j.jmb.2005.03.083

    View details for Web of Science ID 000229401700017

    View details for PubMedID 15896350

  • Fold recognition aided by constraints from small angle X-ray scattering data PROTEIN ENGINEERING DESIGN & SELECTION Zheng, W. J., Doniach, S. 2005; 18 (5): 209-219

    Abstract

    We performed a systematic exploration of the use of structural information derived from small angle X-ray scattering (SAXS) measurements to improve fold recognition. SAXS data provide the Fourier transform of the histogram of atomic pair distances (pair distribution function) for a given protein and hence can serve as a structural constraint on methods used to determine the native conformational fold of the protein. Here we used it to construct a similarity-based fitness score with which to evaluate candidate structures generated by a threading procedure. In order to combine the SAXS scores with the standard energy scores and other 1D profile-based scores used in threading, we made use both of a linear regression method and of a neural network-based technique to obtain optimal combined fitness scores and applied them to the ranking of candidate structures. Our results show that the use of SAXS data with gapless threading significantly improves the performance of fold recognition.

    View details for DOI 10.1093/protein/gzi026

    View details for Web of Science ID 000229699600001

    View details for PubMedID 15845555

  • Probing counterion modulated repulsion and attraction between nucleic acid duplexes in solution PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bai, Y., Das, R., Millett, I. S., Herschlag, D., Doniach, S. 2005; 102 (4): 1035-1040

    Abstract

    Understanding biological and physical processes involving nucleic acids, such as the binding of proteins to DNA and RNA, DNA condensation, and RNA folding, requires an understanding of the ion atmosphere that surrounds nucleic acids. We have used a simple model DNA system to determine how the ion atmosphere modulates interactions between duplexes in the absence of specific metal ion-binding sites and other complicated interactions. In particular, we have tested whether the Coulomb repulsion between nucleic acids can be reversed by counterions to give a net attraction, as has been proposed recently for the rapid collapse observed early in RNA folding. The conformation of two DNA duplexes tethered by a flexible neutral linker was determined in the presence of a series of cations by small angle x-ray scattering. The small angle x-ray scattering profiles of two control molecules with distinct shapes (a continuous duplex and a mimic of the compact DNA) were in good agreement with predictions, establishing the applicability of this approach. Under low-salt conditions (20 mM Na+), the tethered duplexes are extended because of a Coulombic repulsion estimated to be 2-5 kT/bp. Addition of high concentrations of Na+ (1.2 M), Mg2+ (0.6 M), and spermidine3+ (75 mM) resulted in electrostatic relaxation to a random state. These results indicate that a counterion-induced attractive force between nucleic acid duplexes is not significant under physiological conditions. An upper limit on the magnitude of the attractive potential under all tested ionic conditions is estimated.

    View details for DOI 10.1073/pnas.0404448102

    View details for Web of Science ID 000226617900016

    View details for PubMedID 15647360

  • A comparative study of motor-protein motions by using a simple elastic-network model PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Zheng, W. J., Doniach, S. 2003; 100 (23): 13253-13258

    Abstract

    In this work, we report on a study of the structure-function relationships for three families of motor proteins, including kinesins, myosins, and F1-ATPases, by using a version of the simple elastic-network model of large-scale protein motions originally proposed by Tirion [Tirion, M. (1996) Phys. Rev. Lett. 77, 1905-1908]. We find a surprising dichotomy between kinesins and the other motor proteins (myosins and F1-ATPase). For the latter, there exist one or two dominant lowest-frequency modes (one for myosin, two for F1-ATPase) obtained from normal-mode analysis of the elastic-network model, which overlap remarkably well with the measured conformational changes derived from pairs of solved crystal structures in different states. Furthermore, we find that the computed global conformational changes induced by the measured deformation of the nucleotide-binding pocket also overlap well with the measured conformational changes, which is consistent with the "nucleotide-binding-induced power-stroke" scenario. In contrast, for kinesins, this simplicity breaks down. Multiple modes are needed to generate the measured conformational changes, and the computed displacements induced by deforming the nucleotide-binding pocket also overlap poorly with the measured conformational changes, and are insufficient to explain the large-scale motion of the relay helix and the linker region. This finding may suggest the presence of two different mechanisms for myosins and kinesins, despite their strong evolutionary ties and structural similarities.

    View details for DOI 10.1073/pnas.2235686100

    View details for Web of Science ID 000186573700027

    View details for PubMedID 14585932

  • The fastest global events in RNA folding: Electrostatic relaxation and tertiary collapse of the tetrahymena ribozyme JOURNAL OF MOLECULAR BIOLOGY Das, R., Kwok, L. W., Millett, I. S., Bai, Y., Mills, T. T., JACOB, J., Maskel, G. S., Seifert, S., Mochrie, S. G., Thiyagarajan, P., Doniach, S., Pollack, L., Herschlag, D. 2003; 332 (2): 311-319

    Abstract

    Large RNAs can collapse into compact conformations well before the stable formation of the tertiary contacts that define their final folds. This study identifies likely physical mechanisms driving these early compaction events in RNA folding. We have employed time-resolved small-angle X-ray scattering to monitor the fastest global shape changes of the Tetrahymena ribozyme under different ionic conditions and with RNA mutations that remove long-range tertiary contacts. A partial collapse in each of the folding time-courses occurs within tens of milliseconds with either monovalent or divalent cations. Combined with comparison to predictions from structural models, this observation suggests a relaxation of the RNA to a more compact but denatured conformational ensemble in response to enhanced electrostatic screening at higher ionic concentrations. Further, the results provide evidence against counterion-correlation-mediated attraction between RNA double helices, a recently proposed model for early collapse. A previous study revealed a second 100 ms phase of collapse to a globular state. Surprisingly, we find that progression to this second early folding intermediate requires RNA sequence motifs that eventually mediate native long-range tertiary interactions, even though these regions of the RNA were observed to be solvent-accessible in previous footprinting studies under similar conditions. These results help delineate an analogy between the early conformational changes in RNA folding and the "burst phase" changes and molten globule formation in protein folding.

    View details for DOI 10.1016/S0022-2836(03)00854-4

    View details for Web of Science ID 000185306700002

    View details for PubMedID 12948483

  • Counterion distribution around DNA probed by solution X-ray scattering PHYSICAL REVIEW LETTERS Das, R., Mills, T. T., Kwok, L. W., Maskel, G. S., Millett, I. S., Doniach, S., Finkelstein, K. D., Herschlag, D., Pollack, L. 2003; 90 (18)

    Abstract

    Counterion atmospheres condensed onto charged biopolymers strongly affect their physical properties and biological functions, but have been difficult to quantify experimentally. Here, monovalent and divalent counterion atmospheres around DNA double helices in solution are probed using small-angle x-ray scattering techniques. Modulation of the ion scattering factors by anomalous (resonant) x-ray scattering and by interchanging ion identities yields direct measurements of the scattering signal due to the spatial correlation of surrounding ions to the DNA. The quality of the data permit, for the first time, quantitative tests of extended counterion distributions calculated from atomic-scale models of biologically relevant molecules.

    View details for DOI 10.1103/PhysRevLett.90.188103

    View details for Web of Science ID 000182823900054

    View details for PubMedID 12786045

  • Closing the folding chamber of the eukaryotic chaperonin requires the transition state of ATP hydrolysis CELL Meyer, A. S., Gillespie, J. R., Walther, D., Millet, I. S., Doniach, S., Frydman, J. 2003; 113 (3): 369-381

    Abstract

    Chaperonins use ATPase cycling to promote conformational changes leading to protein folding. The prokaryotic chaperonin GroEL requires a cofactor, GroES, which serves as a "lid" enclosing substrates in the central cavity and confers an asymmetry on GroEL required for cooperative transitions driving the reaction. The eukaryotic chaperonin TRiC/CCT does not have such a cofactor but appears to have a "built-in" lid. Whether this seemingly symmetric chaperonin also operates through an asymmetric cycle is unclear. We show that unlike GroEL, TRiC does not close its lid upon nucleotide binding, but instead responds to the trigonal-bipyramidal transition state of ATP hydrolysis. Further, nucleotide analogs inducing this transition state confer an asymmetric conformation on TRiC. Similar to GroEL, lid closure in TRiC confines the substrates in the cavity and is essential for folding. Understanding the distinct mechanisms governing eukaryotic and bacterial chaperonin function may reveal how TRiC has evolved to fold specific eukaryotic proteins.

    View details for Web of Science ID 000182640800011

    View details for PubMedID 12732144

  • Rapid compaction during RNA folding PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Russell, R., Millettt, I. S., Tate, M. W., Kwok, L. W., Nakatani, B., Gruner, S. M., Mochrie, S. G., Pande, V., Doniach, S., Herschlag, D., Pollack, L. 2002; 99 (7): 4266-4271

    Abstract

    We have used small angle x-ray scattering and computer simulations with a coarse-grained model to provide a time-resolved picture of the global folding process of the Tetrahymena group I RNA over a time window of more than five orders of magnitude. A substantial phase of compaction is observed on the low millisecond timescale, and the overall compaction and global shape changes are largely complete within one second, earlier than any known tertiary contacts are formed. This finding indicates that the RNA forms a nonspecifically collapsed intermediate and then searches for its tertiary contacts within a highly restricted subset of conformational space. The collapsed intermediate early in folding of this RNA is grossly akin to molten globule intermediates in protein folding.

    View details for DOI 10.1073/pnas.072589599

    View details for Web of Science ID 000174856000027

    View details for PubMedID 11929997

  • Protein structure prediction constrained by solution X-ray scattering data and structural homology identification JOURNAL OF MOLECULAR BIOLOGY Zheng, W. J., Doniach, S. 2002; 316 (1): 173-187

    Abstract

    Here we perform a systematic exploration of the use of distance constraints derived from small angle X-ray scattering (SAXS) measurements to filter candidate protein structures for the purpose of protein structure prediction. This is an intrinsically more complex task than that of applying distance constraints derived from NMR data where the identity of the pair of amino acid residues subject to a given distance constraint is known. SAXS, on the other hand, yields a histogram of pair distances (pair distribution function), but the identities of the pairs contributing to a given bin of the histogram are not known. Our study is based on an extension of the Levitt-Hinds coarse grained approach to ab initio protein structure prediction to generate a candidate set of C(alpha) backbones. In spite of the lack of specific residue information inherent in the SAXS data, our study shows that the implementation of a SAXS filter is capable of effectively purifying the set of native structure candidates and thus provides a substantial improvement in the reliability of protein structure prediction. We test the quality of our predicted C(alpha) backbones by doing structural homology searches against the Dali domain library, and find that the results are very encouraging. In spite of the lack of local structural details and limited modeling accuracy at the C(alpha) backbone level, we find that useful information about fold classification can be extracted from this procedure. This approach thus provides a way to use a SAXS data based structure prediction algorithm to generate potential structural homologies in cases where lack of sequence homology prevents identification of candidate folds for a given protein. Thus our approach has the potential to help in determination of the biological function of a protein based on structural homology instead of sequence homology.

    View details for DOI 10.1006/jmbi.2001.5324

    View details for Web of Science ID 000174025900014

    View details for PubMedID 11829511

  • Equilibrium collapse and the kinetic 'foldability' of proteins BIOCHEMISTRY Millet, I. S., Townsley, L. E., Chiti, F., Doniach, S., Plaxco, K. W. 2002; 41 (1): 321-325

    Abstract

    An important element of protein folding theory has been the identification of equilibrium parameters that might uniquely distinguish rapidly folding polypeptide sequences from those that fold slowly. One such parameter, termed sigma, is a dimensionless, equilibrium measure of the coincidence of chain compaction and folding that is predicted to be an important determinant of relative folding kinetics. To test this prediction and improve our understanding of the putative relationship between nonspecific compaction of the unfolded state and protein folding kinetics, we have used small-angle X-ray scattering and circular dichroism spectroscopy to measure the sigma of five well-characterized proteins. Consistent with theoretical predictions, we find that near-perfect coincidence of the unfolded state contraction and folding (sigma approximately 0) is associated with the rapid kinetics of these naturally occurring proteins. We do not, however, observe any significant correlation between sigma and either the relative folding rates of these proteins or the presence or absence of well-populated kinetic intermediates. Thus, while sigma approximately 0 may be a necessary condition to ensure rapid folding, differences in sigma do not account for the wide range of rates and mechanisms with which naturally occurring proteins fold.

    View details for DOI 10.1021/bi015695a

    View details for Web of Science ID 000173216500036

    View details for PubMedID 11772031

  • Exploring the folding landscape of a structured RNA PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Russell, R., Zhuang, X. W., Babcock, H. P., Millett, I. S., Doniach, S., Chu, S., Herschlag, D. 2002; 99 (1): 155-160

    Abstract

    Structured RNAs achieve their active states by traversing complex, multidimensional energetic landscapes. Here we probe the folding landscape of the Tetrahymena ribozyme by using a powerful approach: the folding of single ribozyme molecules is followed beginning from distinct regions of the folding landscape. The experiments, combined with small-angle x-ray scattering results, show that the landscape contains discrete folding pathways. These pathways are separated by large free-energy barriers that prevent interconversion between them, indicating that the pathways lie in deep channels in the folding landscape. Chemical protection and mutagenesis experiments are then used to elucidate the structural features that determine which folding pathway is followed. Strikingly, a specific long-range tertiary contact can either help folding or hinder folding, depending on when it is formed during the process. Together these results provide an unprecedented view of the topology of an RNA folding landscape and the RNA structural features that underlie this multidimensional landscape.

    View details for Web of Science ID 000173233300031

    View details for PubMedID 11756689

  • Toward a taxonomy of the denatured state: Small angle scattering studies of unfolded proteins UNFOLDED PROTEINS Millet, I. S., Doniach, S., Plaxco, K. W. 2002; 62: 241-262

    View details for Web of Science ID 000179263800007

    View details for PubMedID 12418105

  • Changes in biomolecular conformation seen by small angle X-ray scattering CHEMICAL REVIEWS Doniach, S. 2001; 101 (6): 1763-1778

    View details for DOI 10.1021/cr990071k

    View details for Web of Science ID 000169375600008

    View details for PubMedID 11709998

  • Solution structural studies and low-resolution model of the Schizosaccharomyces pombe sap1 protein JOURNAL OF MOLECULAR BIOLOGY Bada, M., Walther, D., Arcangioli, B., Doniach, S., Delarue, M. 2000; 300 (3): 563-574

    Abstract

    Sap1 is a DNA-binding protein involved in controlling the mating type switch in fission yeast Schizosaccharomyces pombe. In the absence of any significant sequence similarity with any structurally known protein, a variety of biophysical techniques has been used to probe the solution low-resolution structure of the sap1 protein. First, sap1 is demonstrated to be an unusually elongated dimer in solution by measuring the translational diffusion coefficient with two independent techniques: dynamic light-scattering and ultracentrifugation. Second, sequence analysis revealed the existence of a long coiled-coil region, which is responsible for dimerization. The length of the predicted coiled-coil matches estimates drawn from the hydrodynamic experimental behaviour of the molecule. In addition, the same measurements done on a shorter construct with a coiled-coil region shortened by roughly one-half confirmed the localization of the long coiled-coil region. A crude T-shape model incorporating all these information was built. Third, small-angle X-ray scattering (SAXS) of the free molecule provided additional evidence for the model. In particular, the P(r) curve strikingly demonstrates the existence of long intramolecular distances. Using a novel 3D reconstruction algorithm, a low resolution 3D model of the protein has been independently constructed that matches the SAXS experimental data. It also fits the translation diffusion coefficients measurements and agrees with the first T-shaped model. This low-resolution model has clearly biologically relevant new functional implications, suggesting that sap1 is a bifunctional protein, with the two active sites being separated by as much as 120 A; a tetrapeptide repeated four times at the C terminus of the molecule is postulated to be of utmost functional importance.

    View details for DOI 10.1006/jmbi.2000.3854

    View details for Web of Science ID 000088223600012

    View details for PubMedID 10884352

  • Small angle X-ray scattering reveals a compact intermediate in RNA folding NATURE STRUCTURAL BIOLOGY Russell, R., Millett, I. S., Doniach, S., Herschlag, D. 2000; 7 (5): 367-370

    Abstract

    We have used small angle X-ray scattering (SAXS) to monitor changes in the overall size and shape of the Tetrahymena ribozyme as it folds. The native ribozyme, formed in the presence of Mg2+, is much more compact and globular than the ensemble of unfolded conformations. Time-resolved measurements show that most of the compaction occurs at least 20-fold faster than the overall folding to the native state, suggesting that a compact intermediate or family of intermediates is formed early and then rearranges in the slow steps that limit the overall folding rate. These results lead to a kinetic folding model in which an initial 'electrostatic collapse' of the RNA is followed by slower rearrangements of elements that are initially mispositioned.

    View details for Web of Science ID 000086908800009

    View details for PubMedID 10802731

  • Fourth-generation X-ray sources: some possible applications to biology JOURNAL OF SYNCHROTRON RADIATION Doniach, S. 2000; 7: 116-120

    Abstract

    The term 'fourth generation X-ray sources' has come to mean X-ray free-electron lasers which use multi-GeV electron beams from linear accelerators to generate X-rays by self-amplified stimulated emission when fired into long undulators. Properties of the radiation expected from such sources are reviewed briefly and two possible applications of the resulting pulsed highly collimated X-radiation to problems in biology are discussed: use of X-ray photon correlation spectroscopy to measure time correlations of atoms in protein crystals, and use of Mössbauer radiation extracted from the photon beams by resonant Bragg diffraction from (57)Fe-containing crystals, for MAD phasing of very large unit-cell biomolecular crystals and possibly for photon echo measurements.

    View details for Web of Science ID 000087250300003

    View details for PubMedID 16609184

  • Transient dimer in the refolding kinetics of cytochrome c characterized by small-angle X-ray scattering BIOCHEMISTRY Segel, D. J., ELIEZER, D., Uversky, V., Fink, A. L., Hodgson, K. O., Doniach, S. 1999; 38 (46): 15352-15359

    Abstract

    The equilibrium unfolding and the kinetic refolding of cytochrome c (Cyt c) in the presence of imidazole were studied with small-angle X-ray scattering (SAXS). The equilibrium unfolding experiments showed the radius of gyration, R(g), of native Cyt c to swell approximately 1 A with the addition of imidazole. The thermodynamic parameter m also reflects an expansion of the protein as its lower value demonstrates an increase in solvent-accessible surface area over that of native Cyt c in the absence of imidazole. Refolding was studied in the presence of imidazole as it prevents misligated intermediate states from forming during the refolding process, simplifying the kinetics, and making them easier to resolve. Time-resolved decreases in the forward scattering amplitude, I(0), demonstrated the transient formation of an aggregated intermediate. Final protein and denaturant concentrations were varied in the refolding kinetics, and the singular value decomposition (SVD) method was employed to characterize the associated state. This state was determined to be a dimer, with properties consistent with a molten globule.

    View details for Web of Science ID 000083899400034

    View details for PubMedID 10563821

  • Characterization of transient intermediates in lysozyme folding with time-resolved small-angle X-ray scattering JOURNAL OF MOLECULAR BIOLOGY Segel, D. J., Bachmann, A., Hofrichter, J., Hodgson, K. O., Doniach, S., Kiefhaber, T. 1999; 288 (3): 489-499

    Abstract

    We have used synchrotron radiation, together with stopped-flow and continuous-flow mixing techniques to monitor refolding of lysozyme at pH 5.2. From data measured at times which range from 14 ms to two seconds, we can monitor changes in the size, the shape and the pair distribution function of the polypeptide chain during the folding process. Comparison of the results with the properties of native and GdmCl-unfolded lysozyme shows that a major chain collapse occurs in the dead-time of mixing. During this process about 50 % of the change in radius of gyration between the unfolded protein and the native state occurs and the polypeptide chain adopts a globular shape. Time-resolved fluorescence spectra of this collapsed state suggest that the hydrophobic side-chains are still highly solvent accessible. A subsequently formed intermediate with helical structure in the alpha-domain is nearly identical in size and shape with native lysozyme and has a solvent-inaccessible hydrophobic core. Despite its native-like properties, this intermediate is only slightly more stable (DeltaG0=-4 kJ/mol) than the collapsed state and still much less stable than native lysozyme (DeltaDeltaG0=36 kJ/mol) at 20 degrees C.

    View details for Web of Science ID 000080204600014

    View details for PubMedID 10329156

  • Protein dynamics simulations from nanoseconds to microseconds CURRENT OPINION IN STRUCTURAL BIOLOGY Doniach, S., Eastman, P. 1999; 9 (2): 157-163

    Abstract

    There have been a number of advances in atomic resolution simulations of biomolecules during the past few years. These have arisen partly from improvements to computer power and partly from algorithmic improvements. There have also been advances in measuring time-dependent fluctuations in proteins using NMR spectroscopy, revealing the importance of fluctuations in the microsecond to millisecond time range. Progress has also been made in measuring how far the simulations are able to represent the accessible phase space that is available to the protein in its native state, in solution, at room temperature. Another area of development is the simulation of protein unfolding at atomic resolution.

    View details for Web of Science ID 000085219800002

    View details for PubMedID 10322213

  • Protein denaturation: A small-angle X-ray scattering study of the ensemble of unfolded states of cytochrome c BIOCHEMISTRY Segel, D. J., Fink, A. L., Hodgson, K. O., Doniach, S. 1998; 37 (36): 12443-12451

    Abstract

    Solution X-ray scattering was used to study the equilibrium unfolding of cytochrome c as a function of guanidine hydrochloride concentration at neutral pH. The radius of gyration (Rg) shows a cooperative transition with increasing denaturant with a similar Cm to that observed with circular dichroism. However, the lack of an isoscattering point in the X-ray scattering patterns suggests the equilibrium unfolding is not simply a two-state process. Singular value decomposition (SVD) analysis was applied to the scattering patterns to determine the number of distinct scattering species. SVD analysis reveals the existence of three components, suggesting that at least three equilibrium states of the protein exist. A model was employed to determine the thermodynamic parameters and the scattering profiles of the three equilibrium states. These scattering profiles show that one state is native (N). The other two states (U1, U2) are unfolded, with U2 being fully unfolded and U1 having some residual structure. Using the thermodynamic parameters to calculate fractional populations, U1 is maximally populated at intermediate denaturant concentrations while U2 is maximally populated at high denaturant concentrations. It is likely that there is a multiplicity of denatured states with U1 and U2 representing an average of the denatured states populated at intermediate and high denaturant concentrations, respectively.

    View details for Web of Science ID 000075909800009

    View details for PubMedID 9730816

  • Multiple time step diffusive Langevin dynamics for proteins PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS Eastman, P., Doniach, S. 1998; 30 (3): 215-227

    Abstract

    We present an algorithm for simulating the long time scale dynamics of proteins and other macromolecules. Our method applies the concept of multiple time step integration to the diffusive Langevin equation, in which short time scale dynamics are replaced by friction and noise. The macromolecular force field is represented at atomic resolution. Slow motions are modeled by constrained Langevin dynamics with very large time steps, while faster degrees of freedom are kept in local thermal equilibrium. In the limit of a sufficiently large molecule, our algorithm is shown to reduce the CPU time required by two orders of magnitude. We test the algorithm on two systems, alanine dipeptide and bovine pancreatic trypsin inhibitor (BPTI), and find that it accurately calculates a variety of equilibrium and dynamical properties. In the case of BPTI, the CPU time required is reduced by nearly a factor of 60 compared to a conventional, unconstrained Langevin simulation using the same force field.

    View details for Web of Science ID 000072331600001

    View details for PubMedID 9517537

  • Kinetics of lysozyme refolding: Structural characterization of a non-specifically collapsed state using time-resolved X-ray scattering JOURNAL OF MOLECULAR BIOLOGY Chen, L. L., Wildegger, G., Kiefhaber, T., Hodgson, K. O., Doniach, S. 1998; 276 (1): 225-237

    Abstract

    We report time-resolved small angle X-ray scattering (SAXS) studies of the structural characteristics of the collapsed state of lysozyme from henegg white (HEL) obtained on initiating refolding by rapidly changing solvent conditions from 8 M to 1.1 M urea at pH 2.9. At this reduced pH the lifetime, of about one second, of the non-specifically collapsed ensemble is considerably prolonged relative to its value at pH 5.2. The SAXS studies are combined with time resolved measurements of tryptophan fluorescence and of the rate of formation of native molecules using interrupted refolding experiments. We observe large burst phase changes in intrinsic tryptophan fluorescence and in the radius of gyration (Rg) which is reduced from 22 A in the fully unfolded state to approximately 19 to 20 A. Subsequent decrease of the Rg to the value for native lysozyme (15 A) follows the time course of formation of native molecules. Single exponential fits to the singular value decomposition (SVD) components of the SAXS data allow reconstruction of the SAXS profile at early time points of refolding. The results of this analysis suggest a globular shape of the collapsed state. A similar fit to the forward scattering amplitude, I(0), suggests that the collapsed state has a solvent accessible surface area which is considerably increased relative to that of the native protein. These results show directly that the non-specifically collapsed state formed during the burst phase in lysozyme refolding indeed represents a molecular compaction and a change in shape from a fully denatured random coil state (albeit restricted by disulfide bonds) to an ensemble of globular conformations which, however, have not yet formed a solvent-protected hydrophobic core.

    View details for Web of Science ID 000072310200016

    View details for PubMedID 9514723

  • A lysozyme folding intermediate revealed by solution X-ray scattering JOURNAL OF MOLECULAR BIOLOGY Chen, L. L., Hodgson, K. O., Doniach, S. 1996; 261 (5): 658-671

    Abstract

    Equilibrium unfolding of hen egg lysozyme as a function of urea concentration at pH 2.9 has been studied by solution X-ray scattering. Differences in the unfolding transition are observed as monitored by the radius of gyration Rg, and by far and near UV CD (circular dichroism) at 222 nm and 298 nm, respectively. This suggests the existence of a third unfolding species, in addition to the native and the unfolded states. A singular value decomposition (SVD) analysis was made of the scattering curves at different urea concentrations. This analysis shows clear evidence of a third basis component in the X-ray scattering curves, thus supporting the results of the Rg and CD measurements. The denaturant binding model was employed to estimate the thermodynamic parameters of denaturation for the intermediate and unfolded states. Use of these parameters to refine the SVD analysis allows us to reconstruct a scattering profile for the pure intermediate state. Simplified partially folded models, based on the crystal structure of hen lysozyme, support a working model for the intermediate, whose structure may be correlated with that of the kinetic intermediate found in the refolding pathway studied by Dobson and coworkers.

    View details for Web of Science ID A1996VF29600007

    View details for PubMedID 8800214

  • BLUE FORM OF BACTERIORHODOPSIN AND ITS ORDER-DISORDER TRANSITION DURING DEHYDRATION BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS Wakatsuki, S., Kimura, Y., Stoeckenius, W., Gillis, N., ELIEZER, D., Hodgson, K. O., Doniach, S. 1994; 1185 (2): 160-166

    Abstract

    Freshly-prepared blue membranes from Halobacterium halobium, previously reported to be disordered, are shown to have a distinct crystal lattice structure, slightly different from the native form. The lattice of the blue form is disrupted irreversibly when dehydrated. The disorder process was observed using time-resolved small-angle X-ray diffraction and analyzed by radial autocorrelation functions. The diffraction peaks of the in-plane lattice first sharpen and increase due to improved membrane orientation, then the trimer lattice becomes disordered and the unit cell dimension decreases by 1.8 A. In contrast, dehydration of purple membranes does not disorder the lattice, and the unit cell dimension shrinks by only 1.0 A. Comparisons of radial autocorrelation functions for the blue membrane during drying show drastic loss of inter-trimer, long-range correlation while the intra-trimer, short-range correlations remain more or less unchanged. This suggests that the deionized protein trimers can maintain their overall structure during the dehydration, even though the lattice dimension decreases appreciably and the two-dimensional crystallinity is disrupted.

    View details for Web of Science ID A1994NK09700003

    View details for PubMedID 8167134

  • MGATP-INDUCED CONFORMATIONAL-CHANGES IN THE IRON PROTEIN FROM AZOTOBACTER-VINELANDII, AS STUDIED BY SMALL-ANGLE X-RAY-SCATTERING JOURNAL OF BIOLOGICAL CHEMISTRY Chen, L., Gavini, N., Tsuruta, H., ELIEZER, D., Burgess, B. K., Doniach, S., Hodgson, K. O. 1994; 269 (5): 3290-3294

    Abstract

    Small angle x-ray scattering experiments have been carried out on the purified iron proteins of nitrogenase from wild-type Azotobacter vinelandii and from a Nif- mutant strain, A. vinelandii UW91 (which has an A157S mutation). This study was designed to investigate the influence of MgATP and MgADP binding on the protein structure in solution. For the wild-type protein, the binding of MgATP induces a significant conformational change that is observed as a decrease of about 2.0 A in the radius of gyration. In contrast, the binding of MgADP to the wild-type iron protein does not detectably affect the radius of gyration. In the absence of nucleotides, the radius of gyration for the UW91 mutant is indistinguishable from that of the wild-type. However, unlike for the wild-type protein, the radius of gyration of the UW91 iron protein is unaffected by the addition of MgATP. We have previously shown that the UW91 iron protein has a normal [4Fe-4S] cluster and MgATP binding ability but that it is completely blocked for electron transfer and MgATP hydrolysis (Gavini, N., and Burgess, B. K. (1992) J. Biol. Chem. 267, 21179-21186). These x-ray scattering measurements suggest that a conformation different from that of the native state is therefore required for the iron protein to perform electron transfer to the MoFe protein. These results also support the hypothesis that Ala-157 is crucial for the iron protein to establish the electron-transfer-favored conformation induced by MgATP binding.

    View details for Web of Science ID A1994MV63100028

    View details for PubMedID 8106367

  • SMALL-ANGLE X-RAY-SCATTERING STUDIES OF THE IRON-MOLYBDENUM COFACTOR FROM AZOTOBACTER-VINELANDII NITROGENASE JOURNAL OF BIOLOGICAL CHEMISTRY ELIEZER, D., Frank, P., Gillis, N., Newton, W. E., Doniach, S., Hodgson, K. O. 1993; 268 (28): 20953-20957

    Abstract

    The nitrogenase enzyme complex, consisting of the molybdenum-iron protein and the iron protein, plays a critical role in the biological reduction of dinitrogen to ammonia (nitrogen fixation). The nitrogen-fixing site within the molybdenum-iron protein is an iron-molybdenum-sulfur cofactor (FeMoco) of roughly 1000-2000 Dalton mass. Structural aspects of FeMoco have been determined by spectroscopic and more recently by crystallographic studies. In order to determine the radius of gyration (Rg) of isolated FeMoco, we have performed small-angle x-ray scattering studies of FeMoco in N-methylformamide solution, in the absence of the molybdenum-iron protein. Model compounds of known structure have also been examined in similar solvents, N,N-dimethylformamide and acetonitrile, as controls and for calibration purposes. The Rg values obtained for the models are in good agreement with calculations based upon their respective crystal structures. However, the Rg obtained for FeMoco clearly indicates that the cofactor is not monomeric in solution, but rather aggregated and possibly polydisperse. Further, Rg values were also measured after addition of thiol, dithionite, and thiol and dithionite, to the FeMoco samples. The results indicate, surprisingly, that oxidation state and putative thiol coordination have no detectable effect on the aggregation behavior of FeMoco in solution, as determined by these measurements.

    View details for Web of Science ID A1993MA28800050

    View details for PubMedID 8407930

  • EVIDENCE OF AN ASSOCIATIVE INTERMEDIATE ON THE MYOGLOBIN REFOLDING PATHWAY BIOPHYSICAL JOURNAL ELIEZER, D., Chiba, K., Tsuruta, H., Doniach, S., Hodgson, K. O., Kihara, H. 1993; 65 (2): 912-917

    Abstract

    Time-resolved small-angle x-ray scattering using the stopped-flow method has been applied successfully to investigate the refolding of myoglobin. This is the only method to date that yields direct information on protein physical dimensions during the folding process. It has the potential to detect and probe important processes, such as protein compaction and association, on a millisecond time scale. Initial experiments were performed with horse myoglobin denatured in high concentrations of urea. The denatured protein was diluted rapidly into a buffer containing no urea or low concentrations of urea. The time-course of the forward-scattered intensity shows a decrease in amplitude which is clearly not engendered by the compaction of the protein, but does correspond well to a dimer dissociation process. Initial and final radii of gyration correspond well to a dimer and a monomer, respectively. Kratky plots of the initial and final states also support the transient dimerization model. The apparent dissociation rate constant was obtainable directly from the data. An association rate constant and an equilibrium constant could be estimated. The dimerizing intermediate is speculated to be a globular non-native state with an exposed hydrophobic surface.

    View details for Web of Science ID A1993LU23300038

    View details for PubMedID 8218914

  • COMPUTER-SIMULATION OF ANTIBODY-BINDING SPECIFICITY PROTEINS-STRUCTURE FUNCTION AND GENETICS Pellegrini, M., Doniach, S. 1993; 15 (4): 436-444

    Abstract

    A Monte Carlo algorithm that searches for the optimal docking configuration of hen egg white lysozyme to an antibody is developed. Both the lysozyme and the antibody are kept rigid. Unlike the work of other authors, our algorithm does not attempt to explicitly maximize surface contact, but minimizes the energy computed using coarse-grained pair potentials. The final refinement of our best solutions using all-atom OPLS potentials (Jorgensen and Tirado-Rives8) consistently yields the native conformation as the preferred solution for three different antibodies. We find that the use of an exponential distance-dependent dielectric function is an improvement over the more commonly used linear form.

    View details for Web of Science ID A1993KT96700009

    View details for PubMedID 8460113

  • SMALL-ANGLE X-RAY-SCATTERING INVESTIGATION OF THE SOLUTION STRUCTURE OF TROPONIN-C JOURNAL OF BIOLOGICAL CHEMISTRY Hubbard, S. R., Hodgson, K. O., Doniach, S. 1988; 263 (9): 4151-4158

    Abstract

    X-ray crystallographic studies of troponin C (Herzberg, O., and James, M.N.G. (1985) Nature 313, 653-659; Sundaralingam, M., Bergstrom, R., Strasburg, G., Rao, S.T., and Roychowdhury, P. (1985a) Science 227, 945-948) have revealed a novel protein structure consisting of two globular domains, each containing two Ca2+-binding sites, connected via a nine-turn alpha-helix, three turns of which are fully exposed to solvent. Since the crystals were grown at pH approximately 5, it is of interest to determine whether this structure is applicable to the protein in solution under physiological conditions. We have used small-angle x-ray scattering to examine the solution structure of troponin C at pH 6.8 and the effect of Ca2+ on the structure. The scattering data are consistent with an elongated structure in solution with a radius of gyration of approximately 23.0 A, which is quite comparable to that computed for the crystal structure. The experimental scattering profile and the scattering profile computed from the crystal structure coordinates do, however, exhibit differences at the 40-A level. A weak Ca2+-facilitated dimerization of troponin C was observed. The data rule out large Ca2+-induced structural changes, indicating rather that the molecule with Ca2+ bound is only slightly more compact than the Ca2+-free molecule.

    View details for Web of Science ID A1988M662000018

    View details for PubMedID 3346242

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