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  • Single-molecule superresolution imaging allows quantitative analysis of RAF multimer formation and signaling PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Nan, X., Collisson, E. A., Lewis, S., Huang, J., Tamgueney, T. M., Liphardt, J. T., McCormick, F., Gray, J. W., Chu, S. 2013; 110 (46): 18519-18524

    Abstract

    The RAF serine/threonine kinases regulate cell growth through the MAPK pathway, and are targeted by small-molecule RAF inhibitors (RAFis) in human cancer. It is now apparent that protein multimers play an important role in RAF activation and tumor response to RAFis. However, the exact stoichiometry and cellular location of these multimers remain unclear because of the lack of technologies to visualize them. In the present work, we demonstrate that photoactivated localization microscopy (PALM), in combination with quantitative spatial analysis, provides sufficient resolution to directly visualize protein multimers in cells. Quantitative PALM imaging showed that CRAF exists predominantly as cytoplasmic monomers under resting conditions but forms dimers as well as trimers and tetramers at the cell membrane in the presence of active RAS. In contrast, N-terminal truncated CRAF (CatC) lacking autoinhibitory domains forms constitutive dimers and occasional tetramers in the cytoplasm, whereas a CatC mutant with a disrupted CRAF-CRAF dimer interface does not. Finally, artificially forcing CRAF to the membrane by fusion to a RAS CAAX motif induces multimer formation but activates RAF/MAPK only if the dimer interface is intact. Together, these quantitative results directly confirm the existence of RAF dimers and potentially higher-order multimers and their involvement in cell signaling, and showed that RAF multimer formation can result from multiple mechanisms and is a critical but not sufficient step for RAF activation.

    View details for DOI 10.1073/pnas.1318188110

    View details for Web of Science ID 000326830900054

    View details for PubMedID 24158481

  • Ultrahigh-resolution imaging reveals formation of neuronal SNARE/Munc18 complexes in situ PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Pertsinidis, A., Mukherjee, K., Sharma, M., Pang, Z. P., Park, S. R., Zhang, Y., Brunger, A. T., Suedhof, T. C., Chu, S. 2013; 110 (30): E2812-E2820

    Abstract

    Membrane fusion is mediated by complexes formed by SNAP-receptor (SNARE) and Secretory 1 (Sec1)/mammalian uncoordinated-18 (Munc18)-like (SM) proteins, but it is unclear when and how these complexes assemble. Here we describe an improved two-color fluorescence nanoscopy technique that can achieve effective resolutions of up to 7.5-nm full width at half maximum (3.2-nm localization precision), limited only by stochastic photon emission from single molecules. We use this technique to dissect the spatial relationships between the neuronal SM protein Munc18-1 and SNARE proteins syntaxin-1 and SNAP-25 (25 kDa synaptosome-associated protein). Strikingly, we observed nanoscale clusters consisting of syntaxin-1 and SNAP-25 that contained associated Munc18-1. Rescue experiments with syntaxin-1 mutants revealed that Munc18-1 recruitment to the plasma membrane depends on the Munc18-1 binding to the N-terminal peptide of syntaxin-1. Our results suggest that in a primary neuron, SNARE/SM protein complexes containing syntaxin-1, SNAP-25, and Munc18-1 are preassembled in microdomains on the presynaptic plasma membrane. Our superresolution imaging method provides a framework for investigating interactions between the synaptic vesicle fusion machinery and other subcellular systems in situ.

    View details for DOI 10.1073/pnas.1310654110

    View details for Web of Science ID 000322112300011

    View details for PubMedID 23821748

  • Studying calcium-triggered vesicle fusion in a single vesicle-vesicle content and lipid-mixing system NATURE PROTOCOLS Kyoung, M., Zhang, Y., Diao, J., Chu, S., Brunger, A. T. 2013; 8 (1): 1-16

    Abstract

    This protocol describes a single vesicle-vesicle microscopy system to study Ca(2+)-triggered vesicle fusion. Donor vesicles contain reconstituted synaptobrevin and synaptotagmin-1. Acceptor vesicles contain reconstituted syntaxin and synaptosomal-associated protein 25 (SNAP-25), and they are tethered to a PEG-coated glass surface. Donor vesicles are mixed with the tethered acceptor vesicles and incubated for several minutes at a zero-Ca(2+) concentration, resulting in a collection of single interacting vesicle pairs. The donor vesicles also contain two spectrally distinct fluorophores that allow simultaneous monitoring of temporal changes of the content and membrane. Upon Ca(2+) injection into the sample chamber, our system therefore differentiates between hemifusion and complete fusion of interacting vesicle pairs and determines the temporal sequence of these events on a sub-100-millisecond time scale. Other factors such as complexin can be easily added. Our system is unique in that it monitors both content and lipid mixing and starts from a metastable state of interacting vesicle pairs before Ca(2+) injection.

    View details for DOI 10.1038/nprot.2012.134

    View details for Web of Science ID 000313051300001

    View details for PubMedID 23222454

  • Synaptic proteins promote calcium-triggered fast transition from point contact to full fusion ELIFE Diao, J., Grob, P., Cipriano, D. J., Kyoung, M., Zhang, Y., Shah, S., Amie Nguyen, A., Padolina, M., Srivastava, A., Vrljic, M., Shah, A., Nogales, E., Chu, S., Brunger, A. T. 2012; 1
  • Synaptic proteins promote calcium-triggered fast transition from point contact to full fusion. eLife Diao, J., Grob, P., Cipriano, D. J., Kyoung, M., Zhang, Y., Shah, S., Nguyen, A., Padolina, M., Srivastava, A., Vrljic, M., Shah, A., Nogales, E., Chu, S., Brunger, A. T. 2012; 1

    Abstract

    The molecular underpinnings of synaptic vesicle fusion for fast neurotransmitter release are still unclear. Here, we used a single vesicle-vesicle system with reconstituted SNARE and synaptotagmin-1 proteoliposomes to decipher the temporal sequence of membrane states upon Ca(2+)-injection at 250-500 ?M on a 100-ms timescale. Furthermore, detailed membrane morphologies were imaged with cryo-electron microscopy before and after Ca(2+)-injection. We discovered a heterogeneous network of immediate and delayed fusion pathways. Remarkably, all instances of Ca(2+)-triggered immediate fusion started from a membrane-membrane point-contact and proceeded to complete fusion without discernible hemifusion intermediates. In contrast, pathways that involved a stable hemifusion diaphragm only resulted in fusion after many seconds, if at all. When complexin was included, the Ca(2+)-triggered fusion network shifted towards the immediate pathway, effectively synchronizing fusion, especially at lower Ca(2+)-concentration. Synaptic proteins may have evolved to select this immediate pathway out of a heterogeneous network of possible membrane fusion pathways.DOI:http://dx.doi.org/10.7554/eLife.00109.001.

    View details for DOI 10.7554/eLife.00109

    View details for PubMedID 23240085

  • Post-Translational Modifications and Lipid Binding Profile of Insect Cell-Expressed Full-Length Mammalian Synaptotagmin 1 BIOCHEMISTRY Vrljic, M., Strop, P., Hill, R. C., Hansen, K. C., Chu, S., Brunger, A. T. 2011; 50 (46): 9998-10012

    Abstract

    Synaptotagmin 1 (Syt1) is a Ca(2+) sensor for SNARE-mediated, Ca(2+)-triggered synaptic vesicle fusion in neurons. It is composed of luminal, transmembrane, linker, and two Ca(2+)-binding (C2) domains. Here we describe expression and purification of full-length mammalian Syt1 in insect cells along with an extensive biochemical characterization of the purified protein. The expressed and purified protein is properly folded and has increased ?-helical content compared to the C2AB fragment alone. Post-translational modifications of Syt1 were analyzed by mass spectrometry, revealing the same modifications of Syt1 that were previously described for Syt1 purified from brain extract or mammalian cell lines, along with a novel modification of Syt1, tyrosine nitration. A lipid binding screen with both full-length Syt1 and the C2AB fragments of Syt1 and Syt3 isoforms revealed new Syt1-lipid interactions. These results suggest a conserved lipid binding mechanism in which Ca(2+)-independent interactions are mediated via a lysine rich region of the C2B domain while Ca(2+)-dependent interactions are mediated via the Ca(2+)-binding loops.

    View details for DOI 10.1021/bi200998y

    View details for Web of Science ID 000296893700007

    View details for PubMedID 21928778

  • In vitro system capable of differentiating fast Ca2+-triggered content mixing from lipid exchange for mechanistic studies of neurotransmitter release PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kyoung, M., Srivastava, A., Zhang, Y., Diao, J., Vrljic, M., Grob, P., Nogales, E., Chu, S., Brunger, A. T. 2011; 108 (29): E304-E313

    Abstract

    Understanding the molecular principles of synaptic vesicle fusion is a long-sought goal. It requires the development of a synthetic system that allows manipulations and observations not possible in vivo. Here, we report an in vitro system with reconstituted synaptic proteins that meets the long-sought goal to produce fast content release in the millisecond time regime upon Ca(2+) triggering. Our system simultaneously monitors both content and lipid exchange, and it starts from stable interacting pairs of donor and acceptor vesicles, mimicking the readily releasable pool of synaptic vesicles prior to an action potential. It differentiates between single-vesicle interaction, hemifusion, and complete fusion, the latter mimicking quantized neurotransmitter release upon exocytosis of synaptic vesicles. Prior to Ca(2+) injection, the system is in a state in which spontaneous fusion events between donor and acceptor vesicles are rare. Upon Ca(2+) injection, a rapid burst of complete fusion events emerges, followed by a biphasic decay. The present study focuses on neuronal SNAREs, the Ca(2+) sensor synaptotagmin 1, and the modulator complexin. However, other synaptic proteins could be added and their function examined. Ca(2+) triggering is cooperative, requiring the presence of synaptotagmin, whereas SNAREs alone do not produce a fast fusion burst. Manipulations of the system mimic effects observed in vivo. These results also show that neuronal SNAREs alone do not efficiently produce complete fusion, that the combination of SNAREs with synaptotagmin lowers the activation barriers to full fusion, and that complexin enhances this kinetic control.

    View details for DOI 10.1073/pnas.1107900108

    View details for Web of Science ID 000292876900007

    View details for PubMedID 21705659

  • Three-dimensional molecular modeling with single molecule FRET JOURNAL OF STRUCTURAL BIOLOGY Brunger, A. T., Strop, P., Vrljic, M., Chu, S., Weninger, K. R. 2011; 173 (3): 497-505

    Abstract

    Single molecule fluorescence energy transfer experiments enable investigations of macromolecular conformation and folding by the introduction of fluorescent dyes at specific sites in the macromolecule. Multiple such experiments can be performed with different labeling site combinations in order to map complex conformational changes or interactions between multiple molecules. Distances that are derived from such experiments can be used for determination of the fluorophore positions by triangulation. When combined with a known structure of the macromolecule(s) to which the fluorophores are attached, a three-dimensional model of the system can be determined. However, care has to be taken to properly derive distance from fluorescence energy transfer efficiency and to recognize the systematic or random errors for this relationship. Here we review the experimental and computational methods used for three-dimensional modeling based on single molecule fluorescence resonance transfer, and describe recent progress in pushing the limits of this approach to macromolecular complexes.

    View details for DOI 10.1016/j.jsb.2010.09.004

    View details for Web of Science ID 000287681200010

    View details for PubMedID 20837146

  • Subnanometre single-molecule localization, registration and distance measurements NATURE Pertsinidis, A., Zhang, Y., Chu, S. 2010; 466 (7306): 647-U11

    Abstract

    Remarkable progress in optical microscopy has been made in the measurement of nanometre distances. If diffraction blurs the image of a point object into an Airy disk with a root-mean-squared (r.m.s.) size of s = 0.44lambda/2NA (approximately 90 nm for light with a wavelength of lambda = 600 nm and an objective lens with a numerical aperture of NA = 1.49), limiting the resolution of the far-field microscope in use to d = 2.4s approximately = 200 nm, additional knowledge about the specimen can be used to great advantage. For example, if the source is known to be two spatially resolved fluorescent molecules, the distance between them is given by the separation of the centres of the two fluorescence images. In high-resolution microwave and optical spectroscopy, there are numerous examples where the line centre is determined with a precision of less than 10(-6) of the linewidth. In contrast, in biological applications the brightest single fluorescent emitters can be detected with a signal-to-noise ratio of approximately 100, limiting the centroid localization precision to s(loc) > or = 1% (> or = 1 nm) of the r.m.s. size, s, of the microscope point spread function (PSF). Moreover, the error in co-localizing two or more single emitters is notably worse, remaining greater than 5-10% (5-10 nm) of the PSF size. Here we report a distance resolution of s(reg) = 0.50 nm (1sigma) and an absolute accuracy of s(distance) = 0.77 nm (1sigma) in a measurement of the separation between differently coloured fluorescent molecules using conventional far-field fluorescence imaging in physiological buffer conditions. The statistical uncertainty in the mean for an ensemble of identical single-molecule samples is limited only by the total number of collected photons, to s(loc) approximately 0.3 nm, which is approximately 3 x 10(-3) times the size of the optical PSF. Our method may also be used to improve the resolution of many subwavelength, far-field imaging methods such as those based on co-localization of molecules that are stochastically switched on in space. The improved resolution will allow the structure of large, multisubunit biological complexes in biologically relevant environments to be deciphered at the single-molecule level.

    View details for DOI 10.1038/nature09163

    View details for Web of Science ID 000280412100058

    View details for PubMedID 20613725

  • Molecular mechanism of the synaptotagmin-SNARE interaction in Ca2+-triggered vesicle fusion NATURE STRUCTURAL & MOLECULAR BIOLOGY Vrljic, M., Strop, P., Ernst, J. A., Sutton, R. B., Chu, S., Brunger, A. T. 2010; 17 (3): 325-U92

    Abstract

    In neurons, SNAREs, synaptotagmin and other factors catalyze Ca(2+)-triggered fusion of vesicles with the plasma membrane. The molecular mechanism of this process, especially the interaction between synaptotagmin and SNAREs, remains an enigma. Here we characterized this interaction by single-molecule fluorescence microscopy and crystallography. The two rigid Ca(2+)-binding domains of synaptotagmin 3 (Syt3) undergo large relative motions in solution. Interaction with SNARE complex amplifies a particular state of the two domains that is further enhanced by Ca(2+). This state is represented by the first SNARE-induced Ca(2+)-bound crystal structure of a synaptotagmin fragment containing both domains. The arrangement of the Ca(2+)-binding loops of this structure of Syt3 matches that of SNARE-bound Syt1, suggesting a conserved feature of synaptotagmins. The loops resemble the membrane-interacting loops of certain viral fusion proteins in the postfusion state, suggesting unexpected similarities between both fusion systems.

    View details for DOI 10.1038/nsmb.1764

    View details for Web of Science ID 000275182700013

    View details for PubMedID 20173762

  • Single-molecule FRET-derived model of the synaptotagmin 1-SNARE fusion complex NATURE STRUCTURAL & MOLECULAR BIOLOGY Choi, U. B., Strop, P., Vrljic, M., Chu, S., Brunger, A. T., Weninger, K. R. 2010; 17 (3): 318-U84

    Abstract

    Synchronous neurotransmission is triggered when Ca(2+) binds to synaptotagmin 1 (Syt1), a synaptic-vesicle protein that interacts with SNAREs and membranes. We used single-molecule fluorescence resonance energy transfer (FRET) between synaptotagmin's two C2 domains to determine that their conformation consists of multiple states with occasional transitions, consistent with domains in random relative motion. SNARE binding results in narrower intrasynaptotagmin FRET distributions and less frequent transitions between states. We obtained an experimentally determined model of the elusive Syt1-SNARE complex using a multibody docking approach with 34 FRET-derived distances as restraints. The Ca(2+)-binding loops point away from the SNARE complex, so they may interact with the same membrane. The loop arrangement is similar to that of the crystal structure of SNARE-induced Ca(2+)-bound Syt3, suggesting a common mechanism by which the interaction between synaptotagmins and SNAREs aids in Ca(2+)-triggered fusion.

    View details for DOI 10.1038/nsmb.1763

    View details for Web of Science ID 000275182700012

    View details for PubMedID 20173763

  • DNA Methylation Increases Nucleosome Compaction and Rigidity JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Choy, J. S., Wei, S., Lee, J. Y., Tan, S., Chu, S., Lee, T. 2010; 132 (6): 1782-?

    Abstract

    Cytosine methylation on CpG dinucleotides is an essential epigenetic modification in eukaryotes. How DNA methylation modulates nucleosome structure and dynamics has been a long-standing question. We implemented a single-molecule method to monitor the effects of DNA methylation on the structure and dynamics of mononucleosomes. Our studies show that DNA methylation induces a more compact and rigid nucleosome structure, providing a physical basis for how DNA methylation might contribute to regulating chromatin structure.

    View details for DOI 10.1021/ja910264z

    View details for Web of Science ID 000275085000025

    View details for PubMedID 20095602

  • Multiple native states reveal persistent ruggedness of an RNA folding landscape NATURE Solomatin, S. V., Greenfeld, M., Chu, S., Herschlag, D. 2010; 463 (7281): 681-U117

    Abstract

    According to the 'thermodynamic hypothesis', the sequence of a biological macromolecule defines its folded, active (or 'native') structure as a global energy minimum in the folding landscape. However, the enormous complexity of folding landscapes of large macromolecules raises the question of whether there is in fact a unique global minimum corresponding to a unique native conformation or whether there are deep local minima corresponding to alternative active conformations. The folding of many proteins is well described by two-state models, leading to highly simplified representations of protein folding landscapes with a single native conformation. Nevertheless, accumulating experimental evidence suggests a more complex topology of folding landscapes with multiple active conformations that can take seconds or longer to interconvert. Here we demonstrate, using single-molecule experiments, that an RNA enzyme folds into multiple distinct native states that interconvert on a timescale much longer than that of catalysis. These data demonstrate that severe ruggedness of RNA folding landscapes extends into conformational space occupied by native conformations.

    View details for DOI 10.1038/nature08717

    View details for Web of Science ID 000274193900040

    View details for PubMedID 20130651

  • Characterizing the Initial Encounter Complex in Cadherin Adhesion STRUCTURE Sivasankar, S., Zhang, Y., Nelson, W. J., Chu, S. 2009; 17 (8): 1075-1081

    Abstract

    Cadherins are Ca(2+)-dependent cell-cell adhesion proteins with an extracellular region of five domains (EC1 to EC5). Adhesion is mediated by "strand swapping" of a conserved tryptophan residue in position 2 between EC1 domains of opposing cadherins, but the formation of this structure is not well understood. Using single-molecule fluorescence resonance energy transfer and single-molecule force measurements with the atomic force microscope, we demonstrate that cadherins initially interact via EC1 domains without swapping tryptophan-2 to form a weak Ca(2+) dependent initial encounter complex that has 25% of the bond strength of a strand-swapped dimer. We suggest that cadherin dimerization proceeds via an induced fit mechanism where the monomers first form a tryptophan-2 independent initial encounter complex and then undergo subsequent conformational changes to form the final strand-swapped dimer.

    View details for DOI 10.1016/j.str.2009.06.012

    View details for Web of Science ID 000269229100008

    View details for PubMedID 19646884

  • Noise-Immune Conjugate Large-Area Atom Interferometers PHYSICAL REVIEW LETTERS Chiow, S., Herrmann, S., Chu, S., Mueller, H. 2009; 103 (5)

    Abstract

    We present a pair of simultaneous conjugate Ramsey-Bordé atom interferometers using large (20variant Planck's over 2pik)-momentum transfer beam splitters, where variant Planck's over 2pik is the photon momentum. Simultaneous operation allows for common-mode rejection of vibrational noise. This allows us to surpass the enclosed space-time area of previous interferometers with a splitting of 20variant Planck's over 2pik by a factor of 2500. Using a splitting of 10variant Planck's over 2pik, we demonstrate a 3.4 ppb resolution in the measurement of the fine structure constant. Examples for applications in tests of fundamental laws of physics are given.

    View details for DOI 10.1103/PhysRevLett.103.050402

    View details for Web of Science ID 000268618300002

    View details for PubMedID 19792468

  • Atom interferometry tests of local Lorentz invariance in gravity and electrodynamics PHYSICAL REVIEW D Chung, K., Chiow, S., Herrmann, S., Chu, S., Mueller, H. 2009; 80 (1)
  • Atom Interferometers with Scalable Enclosed Area PHYSICAL REVIEW LETTERS Mueller, H., Chiow, S., Herrmann, S., Chu, S. 2009; 102 (24)

    Abstract

    Bloch oscillations (i.e., coherent acceleration of matter waves by an optical lattice) and Bragg diffraction are integrated into light-pulse atom interferometers with large momentum splitting between the interferometer arms, and hence enhanced sensitivity. Simultaneous acceleration of both arms in the same internal states suppresses systematic effects, and simultaneously running a pair of interferometers suppresses the effect of vibrations. Ramsey-Bordé interferometers using four such Bloch-Bragg-Bloch beam splitters exhibit 15% contrast at 24variant Planck's over 2pik splitting, the largest so far (variant Planck's over 2pik is the photon momentum); single beam splitters achieve 88variant Planck's over 2pik. The prospects for reaching 100 s of variant Planck's over 2pik and applications such as gravitational wave sensors are discussed.

    View details for DOI 10.1103/PhysRevLett.102.240403

    View details for Web of Science ID 000267197900003

    View details for PubMedID 19658985

  • Nanoparticle-Mediated Nonfluorescent Bonding of Microspheres to Atomic Force Microscope Cantilevers and Imaging Fluorescence from Bonded Cantilevers with Single Molecule Sensitivity NANO LETTERS Sivasankar, S., Chu, S. 2009; 9 (5): 2120-2124

    Abstract

    A technique to attach silica and glass microspheres onto silicon or silicon nitride cantilevers using silica nanoparticle sol-gel chemistry is presented and a method to image the fluorescence background from the bonded cantilevers with single molecule sensitivity is described. The silica nanoparticles polymerize to form a highly branched network that covalently link the microsphere and cantilever together. The bonding is carried out at room temperature which preserves the integrity of the cantilevers and their reflective coating. Comparison of cantilever and single dye molecule fluorescence demonstrates that the cantilevers are nonfluorescent at the single molecule level.

    View details for DOI 10.1021/nl900616y

    View details for Web of Science ID 000266157100070

    View details for PubMedID 19435383

  • 6 W, 1 kHz linewidth, tunable continuous-wave near-infrared laser OPTICS EXPRESS Chiow, S., Herrmann, S., Mueller, H., Chu, S. 2009; 17 (7): 5246-5250

    Abstract

    A modified Coherent 899-21 titanium sapphire laser is injection locked to produce 6-6.5 W of single-frequency light at 852 nm. After closed-loop amplitude control and frequency stabilization to a high-finesse cavity, it delivers 4-4.5 W with < 1 kHz linewidth at the output of a single-mode fiber. The laser is tunable from about 700-1000 nm; up to 8 W should be possible at 750-810 nm.

    View details for Web of Science ID 000264747500038

    View details for PubMedID 19333288

  • Resolving cadherin interactions and binding cooperativity at the single-molecule level PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Zhang, Y., Sivasankar, S., Nelson, W. J., Chu, S. 2009; 106 (1): 109-114

    Abstract

    The cadherin family of Ca(2+)-dependent cell adhesion proteins are critical for the morphogenesis and functional organization of tissues in multicellular organisms, but the molecular interactions between cadherins that are at the core of cell-cell adhesion are a matter of considerable debate. A widely-accepted model is that cadherins adhere in 3 stages. First, the functional unit of cadherin adhesion is a cis dimer formed by the binding of the extracellular regions of 2 cadherins on the same cell surface. Second, formation of low-affinity trans interactions between cadherin cis dimers on opposing cell surfaces initiates cell-cell adhesion. Third, lateral clustering of cadherins cooperatively strengthens intercellular adhesion. Evidence of these cadherin binding states during adhesion is, however, contradictory, and evidence for cooperativity is lacking. We used single-molecule structural (fluorescence resonance energy transfer) and functional (atomic force microscopy) assays to demonstrate directly that cadherin monomers interact via their N-terminal EC1 domain to form trans adhesive complexes. We could not detect the formation of cadherin cis dimers, but found that increasing the density of cadherin monomers cooperatively increased the probability of trans adhesive binding.

    View details for DOI 10.1073/pnas.0811350106

    View details for Web of Science ID 000262263900023

    View details for PubMedID 19114658

  • Single-Molecule Studies of the Neuronal SNARE Fusion Machinery ANNUAL REVIEW OF BIOCHEMISTRY Brunger, A. T., Weninger, K., Bowen, M., Chu, S. 2009; 78: 903-928

    Abstract

    SNAREs are essential components of the machinery for Ca(2+)-triggered fusion of synaptic vesicles with the plasma membrane, resulting in neurotransmitter release into the synaptic cleft. Although much is known about their biophysical and structural properties and their interactions with accessory proteins such as the Ca(2+) sensor synaptotagmin, their precise role in membrane fusion remains an enigma. Ensemble studies of liposomes with reconstituted SNAREs have demonstrated that SNAREs and accessory proteins can trigger lipid mixing/fusion, but the inability to study individual fusion events has precluded molecular insights into the fusion process. Thus, this field is ripe for studies with single-molecule methodology. In this review, we discuss applications of single-molecule approaches to observe reconstituted SNAREs, their complexes, associated proteins, and their effect on biological membranes. Some of the findings are provocative, such as the possibility of parallel and antiparallel SNARE complexes or of vesicle docking with only syntaxin and synaptobrevin, but have been confirmed by other experiments.

    View details for DOI 10.1146/annurev.biochem.77.070306.103621

    View details for Web of Science ID 000268069200032

    View details for PubMedID 19489736

  • Direct measurement of tertiary contact cooperativity in RNA folding JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Sattint, B. D., Zhao, W., Travers, K., Chut, S., Herschlag, D. 2008; 130 (19): 6085-?

    Abstract

    All structured biological macromolecules must overcome the thermodynamic folding problem to populate a unique functional state among a vast ensemble of unfolded and alternate conformations. The exploration of cooperativity in protein folding has helped reveal and distinguish the underlying mechanistic solutions to this folding problem. Analogous dissections of RNA tertiary stability remain elusive, however, despite the central biological importance of folded RNA molecules and the potential to reveal fundamental properties of structured macromolecules via comparisons of protein and RNA folding. We report a direct quantitative measure of tertiary contact cooperativity in a folded RNA. We precisely measured the stability of an independently folding P4-P6 domain from the Tetrahymena thermophila group I intron by single molecule fluorescence resonance energy transfer (smFRET). Using wild-type and mutant RNAs, we found that cooperativity between the two tertiary contacts enhances P4-P6 stability by 3.2 +/- 0.2 kcal/mol.

    View details for DOI 10.1021/ja800919q

    View details for Web of Science ID 000255620200014

    View details for PubMedID 18429611

  • Atom interferometry with up to 24-photon-momentum-transfer beam splitters PHYSICAL REVIEW LETTERS Mueller, H., Chiow, S., Long, Q., Herrmann, S., Chu, S. 2008; 100 (18)

    Abstract

    We present up to 24-photon Bragg diffraction as a beam splitter in light-pulse atom interferometers to achieve the largest splitting in momentum space so far. Relative to the 2-photon processes used in the most sensitive present interferometers, these large momentum transfer beam splitters increase the phase shift 12-fold for Mach-Zehnder (MZ) and 144-fold for Ramsey-Bordé (RB) geometries. We achieve a high visibility of the interference fringes (up to 52% for MZ or 36% for RB) and long pulse separation times that are possible only in atomic fountain setups. As the atom's internal state is not changed, important systematic effects can cancel.

    View details for DOI 10.1103/PhysRevLett.100.180405

    View details for Web of Science ID 000255771400005

    View details for PubMedID 18518355

  • Atom-wave diffraction between the Raman-Nath and the Bragg regime: Effective Rabi frequency, losses, and phase shifts PHYSICAL REVIEW A Mueller, H., Chiow, S., Chu, S. 2008; 77 (2)
  • Accessory proteins stabilize the acceptor complex for synaptobrevin, the 1 : 1 syntaxin/SNAP-25 complex STRUCTURE Weninger, K., Bowen, M. E., Choi, U. B., Chu, S., Brunger, A. T. 2008; 16 (2): 308-320

    Abstract

    Syntaxin/SNAP-25 interactions precede assembly of the ternary SNARE complex that is essential for neurotransmitter release. This binary complex has been difficult to characterize by bulk methods because of the prevalence of a 2:1 dead-end species. Here, using single-molecule fluorescence, we find the structure of the 1:1 syntaxin/SNAP-25 binary complex is variable, with states changing on the second timescale. One state corresponds to a parallel three-helix bundle, whereas other states show one of the SNAP-25 SNARE domains dissociated. Adding synaptobrevin suppresses the dissociated helix states. Remarkably, upon addition of complexin, Munc13, Munc18, or synaptotagmin, a similar effect is observed. Thus, the 1:1 binary complex is a dynamic acceptor for synaptobrevin binding, and accessory proteins stabilize this acceptor. In the cellular environment the binary complex is actively maintained in a configuration where it can rapidly interact with synaptobrevin, so formation is not likely a limiting step for neurotransmitter release.

    View details for DOI 10.1016/j.str.2007.12.010

    View details for Web of Science ID 000253219400015

    View details for PubMedID 18275821

  • Atom-interferometry tests of the isotropy of post-Newtonian gravity PHYSICAL REVIEW LETTERS Mueller, H., Chiow, S., Herrmann, S., Chu, S., Chung, K. 2008; 100 (3)

    Abstract

    We present a test of the local Lorentz invariance of post-Newtonian gravity by monitoring Earth's gravity with a Mach-Zehnder atom interferometer that features a resolution of up to 8 x 10{-9}g/sqrt[Hz], the highest reported thus far. Expressed within the standard model extension (SME) or Nordtvedt's anisotropic universe model, the analysis limits four coefficients describing anisotropic gravity at the ppb level and three others, for the first time, at the 10 ppm level. Using the SME we explicitly demonstrate how the experiment actually compares the isotropy of gravity and electromagnetism.

    View details for DOI 10.1103/PhysRevLett.100.031101

    View details for Web of Science ID 000252698100013

    View details for PubMedID 18232958

  • Multiphoton- and simultaneous conjugate Ramsey-Borde atom interferometers RECENT DEVELOPMENTS IN GRAVITATION AND COSMOLOGY Mueller, H., Chiow, S., Herrmann, S., Chu, S. 2008; 977: 291-301
  • Nanosecond electro-optical switching with a repetition rate above 20 MHz REVIEW OF SCIENTIFIC INSTRUMENTS Mueller, H., Chiow, S., Herrmann, S., Chu, S. 2007; 78 (12)

    Abstract

    We describe an electro-optical switch based on a commercial electro-optic modulator (modified for high-speed operation) and a 340 V pulser having a rise time of 2.2 ns (at 250 V). It can produce arbitrary pulse patterns with an average repetition rate beyond 20 MHz. It uses a grounded-grid triode driven by transmitting power transistors. We discuss variations that enable analog operation, use the step-recovery effect in bipolar transistors, or offer other combinations of output voltage, size, and cost.

    View details for DOI 10.1063/1.2822101

    View details for Web of Science ID 000251988300030

    View details for PubMedID 18163741

  • Thiostrepton inhibition of tRNA delivery to the ribosome RNA-A PUBLICATION OF THE RNA SOCIETY Gonzalez, R. L., Chu, S., Puglisi, J. D. 2007; 13 (12): 2091-2097

    Abstract

    Ribosome-stimulated hydrolysis of guanosine-5'-triphosphate (GTP) by guanosine triphosphatase (GTPase) translation factors drives protein synthesis by the ribosome. Allosteric coupling of GTP hydrolysis by elongation factor Tu (EF-Tu) at the ribosomal GTPase center to messenger RNA (mRNA) codon:aminoacyl-transfer RNA (aa-tRNA) anticodon recognition at the ribosomal decoding site is essential for accurate and rapid aa-tRNA selection. Here we use single-molecule methods to investigate the mechanism of action of the antibiotic thiostrepton and show that the GTPase center of the ribosome has at least two discrete functions during aa-tRNA selection: binding of EF-Tu(GTP) and stimulation of GTP hydrolysis by the factor. We separate these two functions of the GTPase center and assign each to distinct, conserved structural regions of the ribosome. The data provide a specific model for the coupling between the decoding site and the GTPase center during aa-tRNA selection as well as a general mechanistic model for ribosome-stimulated GTP hydrolysis by GTPase translation factors.

    View details for DOI 10.1261/rna.499407

    View details for Web of Science ID 000250957700005

    View details for PubMedID 17951333

  • Extended-cavity diode lasers with tracked resonances APPLIED OPTICS Chiow, S., Long, Q., Vo, C., Mueller, H., Chu, S. 2007; 46 (33): 7997-8001

    Abstract

    We present a painless, almost-free upgrade to present extended-cavity diode lasers (ECDLs) that improves the long-term mode-hop-free performance by stabilizing the resonance of the internal cavity to the external cavity. This stabilization is based on the observation that the frequency or amplitude noise of the ECDL is lowest at the optimum laser diode temperature or injection current. Thus, keeping the diode current at the level where the noise is lowest ensures mode-hop-free operation within one of the stable regions of the mode chart, even if these should drift due to external influences. This method can be applied directly to existing laser systems without modifying the optical setup. We demonstrate the method in two ECDLs stabilized to vapor cells at 852 and 895 nm wavelengths. We achieve long-term mode-hop-free operation and low noise at low power consumption, even with an inexpensive non-antireflection-coated diode.

    View details for Web of Science ID 000251830400005

    View details for PubMedID 18026536

  • Fluctuations of transfer RNAs between classical and hybrid states BIOPHYSICAL JOURNAL Kim, H. D., Puglisi, J. D., Chu, S. 2007; 93 (10): 3575-3582

    Abstract

    Adjacent transfer RNAs (tRNAs) in the A- and P-sites of the ribosome are in dynamic equilibrium between two different conformations called classical and hybrid states before translocation. Here, we have used single-molecule fluorescence resonance energy transfer to study the effect of Mg(2+) on tRNA dynamics with and without an acetyl group on the A-site tRNA. When the A-site tRNA is not acetylated, tRNA dynamics do not depend on [Mg(2+)], indicating that the relative positions of the substrates for peptide-bond formation are not affected by Mg(2+). In sharp contrast, when the A-site tRNA is acetylated, Mg(2+) lengthens the lifetime of the classical state but does not change the lifetime of the hybrid state. Based on these findings, the classical state resembles a state with direct stabilization of tertiary structure by Mg(2+) ions whereas the hybrid state resembles a state with little Mg(2+)-assisted stabilization. The antibiotic viomycin, a translocation inhibitor, suppresses tRNA dynamics, suggesting that the enhanced fluctuations of tRNAs after peptide-bond formation drive spontaneous attempts at translocation by the ribosome.

    View details for DOI 10.1529/biophysj.107.109884

    View details for Web of Science ID 000250577700023

    View details for PubMedID 17693476

  • A functional dynein-microtubule network is required for NGF signaling through the Rap1/MAPK pathway TRAFFIC Wu, C., Ramirez, A., Cui, B., Ding, J., Delcroix, J. M., Valletta, J. S., Liu, J., Yang, Y., Chu, S., Mobley, W. C. 2007; 8 (11): 1503-1520

    Abstract

    Rap1 transduces nerve growth factor (NGF)/tyrosine receptor kinase A (TrkA) signaling in early endosomes, leading to sustained activation of the p44/p42 mitogen-activated protein kinases (MAPK1/2). However, the mechanisms by which NGF, TrkA and Rap1 are trafficked to early endosomes are poorly defined. We investigated trafficking and signaling of NGF, TrkA and Rap1 in PC12 cells and in cultured rat dorsal root ganglion (DRG) neurons. Herein, we show a role for both microtubule- and dynein-based transport in NGF signaling through MAPK1/2. NGF treatment resulted in trafficking of NGF, TrkA and Rap1 to early endosomes in the perinuclear region of PC12 cells where sustained activation of MAPK1/2 was observed. Disruption of microtubules with nocodazole in PC12 cells had no effect on the activation of TrkA and Ras. However, it disrupted intracellular trafficking of TrkA and Rap1. Moreover, NGF-induced activation of Rap1 and sustained activation of MAPK1/2 were markedly suppressed. Inhibition of dynein activity through overexpression of dynamitin (p50) blocked trafficking of Rap1 and the sustained phase of MAPK1/2 activation in PC12 cells. Remarkably, even in the continued presence of NGF, mature DRG neurons that overexpressed p50 became atrophic and most (>80%) developing DRG neurons died. Dynein- and microtubule-based transport is thus necessary for TrkA signaling to Rap1 and MAPK1/2.

    View details for DOI 10.1111/j.1600-0854.2007.00636.x

    View details for Web of Science ID 000250363800005

    View details for PubMedID 17822405

  • Optical bonding using silica nanoparticle sol-gel chemistry NANO LETTERS Sivasankar, S., Chu, S. 2007; 7 (10): 3031-3034

    Abstract

    A simple method is described to bond optical components using silica nanoparticle sol-gel chemistry. The silica nanoparticles polymerize into highly branched networks that link the surfaces together. The nanoparticle mediated bonding has several advantages to currently used optical joining technologies. The bonding is a room-temperature process and does not require any clean room facilities. The bonded interface has a high mechanical strength and low scattering. The bonding is resistant to organic solvents on silylation with hydrophobic surface groups. This method achieves 100% successful bonding rates between soda-lime glass slides. The bond-setting time can be tailored to allow time for precision optical alignment.

    View details for DOI 10.1021/nl071492h

    View details for Web of Science ID 000250143400017

    View details for PubMedID 17854226

  • One at a time, live tracking of NGF axonal transport using quantum dots PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Cui, B., Wu, C., Chen, L., Ramirez, A., Bearer, E. L., Li, W., Mobley, W. C., Chu, S. 2007; 104 (34): 13666-13671

    Abstract

    Retrograde axonal transport of nerve growth factor (NGF) signals is critical for the survival, differentiation, and maintenance of peripheral sympathetic and sensory neurons and basal forebrain cholinergic neurons. However, the mechanisms by which the NGF signal is propagated from the axon terminal to the cell body are yet to be fully elucidated. To gain insight into the mechanisms, we used quantum dot-labeled NGF (QD-NGF) to track the movement of NGF in real time in compartmentalized culture of rat dorsal root ganglion (DRG) neurons. Our studies showed that active transport of NGF within the axons was characterized by rapid, unidirectional movements interrupted by frequent pauses. Almost all movements were retrograde, but short-distance anterograde movements were occasionally observed. Surprisingly, quantitative analysis at the single molecule level demonstrated that the majority of NGF-containing endosomes contained only a single NGF dimer. Electron microscopic analysis of axonal vesicles carrying QD-NGF confirmed this finding. The majority of QD-NGF was found to localize in vesicles 50-150 nm in diameter with a single lumen and no visible intralumenal membranous components. Our findings point to the possibility that a single NGF dimer is sufficient to sustain signaling during retrograde axonal transport to the cell body.

    View details for DOI 10.1073/pnas.0706192104

    View details for Web of Science ID 000249064700027

    View details for PubMedID 17698956

  • The role of fluctuations in tRNA selection by the ribosome PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lee, T., Blanchard, S. C., Kim, H. D., Puglisi, J. D., Chu, S. 2007; 104 (34): 13661-13665

    Abstract

    The detailed mechanism of how the ribosome decodes protein sequence information with an abnormally high accuracy, after 40 years of study, remains elusive. A critical element in selecting correct transfer RNA (tRNA) transferring correct amino acid is "induced fit" between the ribosome and tRNA. By using single-molecule methods, the induced fit mechanism is shown to position favorably the correct tRNA after initial codon recognition. We provide evidence that this difference in positioning and thermal fluctuations constitutes the primary mechanism for the initial selection of tRNA. This work demonstrates thermal fluctuations playing a critical role in the substrate selection by an enzyme.

    View details for DOI 10.1073/pnas.0705988104

    View details for Web of Science ID 000249064700026

    View details for PubMedID 17699629

  • Measuring the folding transition time of single RNA molecules BIOPHYSICAL JOURNAL Lee, T., Lapidus, L. J., Zhao, W., Travers, K. J., Herschlag, D., Chu, S. 2007; 92 (9): 3275-3283

    Abstract

    We describe a new, time-apertured photon correlation method for resolving the transition time between two states of RNA in folding--i.e., the time of the transition between states rather than the time spent in each state. Single molecule fluorescence resonance energy transfer and fluorescence correlation spectroscopy are used to obtain these measurements. Individual RNA molecules are labeled with fluorophores such as Cy3 and Cy5. Those molecules are then immobilized on a surface and observed for many seconds during which time the molecules spontaneously switch between two conformational states with different levels of flourescence resonance energy transfer efficiency. Single photons are counted from each fluorophore and cross correlated in a small window around a transition. The average of over 1000 cross correlations can be fit to a polynomial, which can determine transition times as short as the average photon emission interval. We applied the method to the P4-P6 domain of the Tetrahymena group I self-splicing intron to yield the folding transition time of 240 micros. The unfolding time is found to be too short to measure with this method.

    View details for DOI 10.1529/biophysj.106.094623

    View details for Web of Science ID 000245544100034

    View details for PubMedID 17307831

  • The individualistic dynamics of entangled DNA in solution MACROMOLECULES Teixeira, R. E., Dambal, A. K., Richter, D. H., Shaqfeh, E. S., Chu, S. 2007; 40 (7): 2461-2476

    View details for DOI 10.1021/ma062932e

    View details for Web of Science ID 000245208400024

  • Peptide bond formation destabilizes Shine-Dalgarno interaction on the ribosome NATURE Uemura, S., Dorywalska, M., Lee, T., Kim, H. D., Puglisi, J. D., Chu, S. 2007; 446 (7134): 454-457

    Abstract

    The ribosome is a molecular machine that translates the genetic code contained in the messenger RNA into an amino acid sequence through repetitive cycles of transfer RNA selection, peptide bond formation and translocation. Here we demonstrate an optical tweezer assay to measure the rupture force between a single ribosome complex and mRNA. The rupture force was compared between ribosome complexes assembled on an mRNA with and without a strong Shine-Dalgarno (SD) sequence-a sequence found just upstream of the coding region of bacterial mRNAs, involved in translation initiation. The removal of the SD sequence significantly reduced the rupture force in complexes carrying an aminoacyl tRNA, Phe-tRNA(Phe), in the A site, indicating that the SD interactions contribute significantly to the stability of the ribosomal complex on the mRNA before peptide bond formation. In contrast, the presence of a peptidyl tRNA analogue, N-acetyl-Phe-tRNA(Phe), in the A site, which mimicked the post-peptidyl transfer state, weakened the rupture force as compared to the complex with Phe-tRNA(Phe), and the resultant force was the same for both the SD-containing and SD-deficient mRNAs. These results suggest that formation of the first peptide bond destabilizes the SD interaction, resulting in the weakening of the force with which the ribosome grips an mRNA. This might be an important requirement to facilitate movement of the ribosome along mRNA during the first translocation step.

    View details for DOI 10.1038/nature05625

    View details for Web of Science ID 000245079500043

    View details for PubMedID 17377584

  • Retrolinkin, a membrane protein, plays an important role in retrograde axonal transport PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Liu, J., Ding, J., Wu, C., Bhagavatula, P., Cui, B., Chu, S., Mobley, W. C., Yang, Y. 2007; 104 (7): 2223-2228

    Abstract

    Retrograde axonal transport plays an important role in the maintenance of neuronal functions, but the mechanism is poorly defined partly because the constituents of the retrograde transport system and their interactions have yet to be elucidated. Of special interest is how dynein/dynactin motor proteins interact with membrane cargoes. Here, we report that an endosomal vesicle protein, termed retrolinkin, functions as a receptor tethering vesicles to dynein/dynactin through BPAG1n4. Retrolinkin, a membrane protein highly enriched in neuronal endosomes, binds directly to BPAG1n4. Deletion of retrolinkin membrane-association domains disrupts retrograde vesicular transport, recapitulating the BPAG1 null phenotype. We propose that retrolinkin acts with BPAG1n4 to specifically regulate retrograde axonal transport. Our work lays the foundation for understanding fundamental issues of axonal transport and provides insights into the molecular mechanisms underlying human neurodegenerative disorders.

    View details for DOI 10.1073/pnas.0602222104

    View details for Web of Science ID 000244438500035

    View details for PubMedID 17287360

  • Gene targeting of GAN in mouse causes a toxic accumulation of microtubule-associated protein 8 and impaired retrograde axonal transport HUMAN MOLECULAR GENETICS Ding, J. Q., Allen, E., Wang, W., Valle, A., Wu, C. B., Nardine, T., Cui, B. X., Yi, J., Taylor, A., Jeon, N. L., Chu, S., So, Y., Vogel, H., Tolwani, R., Mobley, W., Yang, Y. M. 2006; 15 (9): 1451-1463

    Abstract

    Mutations in gigaxonin were identified in giant axonal neuropathy (GAN), an autosomal recessive disorder. To understand how disruption of gigaxonin's function leads to neurodegeneration, we ablated the gene expression in mice using traditional gene targeting approach. Progressive neurological phenotypes and pathological lesions that developed in the GAN null mice recapitulate characteristic human GAN features. The disruption of gigaxonin results in an impaired ubiquitin-proteasome system leading to a substantial accumulation of a novel microtubule-associated protein, MAP8, in the null mutants. Accumulated MAP8 alters the microtubule network, traps dynein motor protein in insoluble structures and leads to neuronal death in cultured wild-type neurons, which replicates the process occurring in GAN null mutants. Defective axonal transport is evidenced by the in vitro assays and is supported by vesicular accumulation in the GAN null neurons. We propose that the axonal transport impairment may be a deleterious consequence of accumulated, toxic MAP8 protein.

    View details for DOI 10.1093/hmg/ddl069

    View details for Web of Science ID 000237002400007

    View details for PubMedID 16565160

  • Phase-locked, low-noise, frequency agile titanium: sapphire lasers for simultaneous atom interferometers OPTICS LETTERS Muller, H., Chiow, S. W., Long, Q., Chu, S. 2006; 31 (2): 202-204

    Abstract

    We demonstrate a laser system consisting of a >1.6 W titanium:sapphire laser that is phase locked to another free-running titanium:sapphire laser at a wavelength of 852 nm with a phase noise of -138 dBc/Hz at 1 MHz from the carrier, using an intracavity electro-optic phase modulator. The residual phase variance is 2.5 x 10(-8) rad2 integrated from 1 Hz to 10 kHz. This system can phase-continuously change the offset frequency within 200 ns with frequency steps up to 4 MHz. Simultaneous atom interferometers can make full use of this ultralow phase noise in differential measurements, where influences from the vibration of optics are greatly suppressed in common mode.

    View details for Web of Science ID 000234665000021

    View details for PubMedID 16441030

  • Active sub-Rayleigh alignment of parallel or antiparallel laser beams OPTICS LETTERS Muller, H., Chiow, S. W., Long, Q., Vo, C., Chu, S. 2005; 30 (24): 3323-3325

    Abstract

    We measure and stabilize the relative angle of parallel and antiparallel laser beams to 5 nrad/(square root of)Hz resolution by comparing the phases of radio frequency beat notes on a quadrant photodetector. The absolute accuracy is 5.1 and 2.1 microrad for antiparallel and parallel beams, respectively, which is more than 6 and 16 times below the Rayleigh criterion.

    View details for Web of Science ID 000233827000021

    View details for PubMedID 16389819

  • Parametric amplification of matter waves in periodically translated optical lattices PHYSICAL REVIEW LETTERS Gemelke, N., Sarajlic, E., Bidel, Y., Hong, S., Chu, S. 2005; 95 (17)

    Abstract

    We observe the sudden growth of small classes of Bloch waves from atomic Bose-Einstein condensates held in periodically translated optical lattices. The effect is explained by narrowband parametric amplification of Bloch waves from noise, due to phase-matched scattering of atom pairs out of the condensate. Amplification occurs above a well-defined modulation threshold, described by dynamic shaping of single-particle band structure.

    View details for DOI 10.1103/PhysRevLett.95.170404

    View details for Web of Science ID 000232724400004

    View details for PubMedID 16383801

  • Phase shifts in precision atom interferometry due to the localization of atoms and optical fields PHYSICAL REVIEW A Wicht, A., Sarajlic, E., Hensley, J. M., Chu, S. 2005; 72 (2)
  • Characteristic periodic motion of polymers in shear flow PHYSICAL REVIEW LETTERS Schroeder, C. M., Teixeira, R. E., Shaqfeh, E. S., Chu, S. 2005; 95 (1)

    Abstract

    The motion of both free and tethered polymer molecules as well as rigid Brownian rods in unbound shear flow is found to be characterized by a clear periodicity or tumbling frequency. Periodicity is shown using a combination of single molecule DNA experiments and computer simulations. In all cases, we develop scaling laws for this behavior and demonstrate that the frequency of characteristic periodic motion scales sublinearly with flow rate.

    View details for DOI 10.1103/PhysRevLett.95.018301

    View details for Web of Science ID 000230275500075

    View details for PubMedID 16090661

  • Single-molecule studies of synaptotagmin and complexin binding to the SNARE complex BIOPHYSICAL JOURNAL Bowen, M. E., Weninger, K., Ernst, J., Chu, S., Brunger, A. T. 2005; 89 (1): 690-702

    Abstract

    The assembly of multiprotein complexes at the membrane interface governs many signaling processes in cells. However, very few methods exist for obtaining biophysical information about protein complex formation at the membrane. We used single molecule fluorescence resonance energy transfer to study complexin and synaptotagmin interactions with the SNARE complex in deposited lipid bilayers. Using total internal reflectance microscopy, individual binding events at the membrane could be resolved despite an excess of unbound protein in solution. Fluorescence resonance energy transfer (FRET)-efficiency derived distances for the complexin-SNARE interaction were consistent with the crystal structure of the complexin-SNARE complex. The unstructured N-terminal region of complexin showed broad distributions of FRET efficiencies to the SNARE complex, suggesting that information on conformational variability can be obtained from FRET efficiency distributions. The low-affinity interaction of synaptotagmin with the SNARE complex changed dramatically upon addition of Ca2+ with high FRET efficiency interactions appearing between the C2B domain and linker domains of synaptotagmin and the membrane proximal portion of the SNARE complex. These results demonstrate that single molecule FRET can be used as a "spectroscopic ruler" to simultaneously gain structural and kinetic information about transient multiprotein complexes at the membrane interface.

    View details for DOI 10.1529/biophysj.104.054064

    View details for Web of Science ID 000230114500067

    View details for PubMedID 15821166

  • Dynamics of DNA in the flow-gradient plane of steady shear flow: Observations and simulations MACROMOLECULES Schroeder, C. M., Teixeira, R. E., Shaqfeh, E. S., Chu, S. 2005; 38 (5): 1967-1978

    View details for DOI 10.1021/ma0480796

    View details for Web of Science ID 000227448200059

  • Shear thinning and tumbling dynamics of single polymers in the flow-gradient plane MACROMOLECULES Teixeira, R. E., Babcock, H. P., Shaqfeh, E. S., Chu, S. 2005; 38 (2): 581-592

    View details for DOI 10.1021/ma0480771

    View details for Web of Science ID 000226466700047

  • Site-specific labeling of the ribosome for single-molecule spectroscopy NUCLEIC ACIDS RESEARCH Dorywalska, M., Blanchard, S. C., Gonzalez, R. L., Kim, H. D., Chu, S., Puglisi, J. D. 2005; 33 (1): 182-189

    Abstract

    Single-molecule fluorescence spectroscopy can reveal mechanistic and kinetic details that may not be observed in static structural and bulk biochemical studies of protein synthesis. One approach requires site-specific and stable attachment of fluorophores to the components of translation machinery. Fluorescent tagging of the ribosome is a prerequisite for the observation of dynamic changes in ribosomal conformation during translation using fluorescence methods. Modifications of the ribosomal particle are difficult due to its complexity and high degree of sequence and structural conservation. We have developed a general method to label specifically the prokaryotic ribosome by hybridization of fluorescent oligonucleotides to mutated ribosomal RNA. Functional, modified ribosomes can be purified as a homogenous population, and fluorescence can be monitored from labeled ribosomal complexes immobilized on a derivatized quartz surface.

    View details for DOI 10.1093/nar/gki151

    View details for Web of Science ID 000226477000017

    View details for PubMedID 15647501

  • Effect of hydrodynamic interactions on DNA dynamics in extensional flow: Simulation and single molecule experiment MACROMOLECULES Schroeder, C. M., Shaqfeh, E. S., Chu, S. 2004; 37 (24): 9242-9256

    View details for DOI 10.1021/ma049461l

    View details for Web of Science ID 000225371200055

  • Single molecule observation of liposome-bilayer fusion thermally induced by soluble N-ethyl maleimide sensitive-factor attachment protein receptors (SNAREs) BIOPHYSICAL JOURNAL Bowen, M. E., Weninger, K., Brunger, A. T., Chu, S. 2004; 87 (5): 3569-3584

    Abstract

    A single molecule fluorescence assay is presented for studying the mechanism of soluble N-ethyl maleimide sensitive-factor attachment protein receptors (SNAREs)-mediated liposome fusion to supported lipid bilayers. The three neuronal SNAREs syntaxin-1A, synaptobrevin-II (VAMP), and SNAP-25A were expressed separately, and various dye-labeled combinations of the SNAREs were tested for their ability to dock liposomes and induce fusion. Syntaxin and synaptobrevin in opposing membranes were both necessary and sufficient to dock liposomes to supported bilayers and to induce thermally activated fusion. As little as one SNARE interaction was sufficient for liposome docking. Fusion of docked liposomes with the supported bilayer was monitored by the dequenching of soluble fluorophores entrapped within the liposomes. Fusion was stimulated by illumination with laser light, and the fusion probability was enhanced by raising the ambient temperature from 22 to 37 degrees C, suggesting a thermally activated process. Surprisingly, SNAP-25 had little effect on docking efficiency or the probability of thermally induced fusion. Interprotein fluorescence resonance energy transfer experiments suggest the presence of other conformational states of the syntaxin*synaptobrevin interaction in addition to those observed in the crystal structure of the SNARE complex. Furthermore, although SNARE complexes involved in liposome docking preferentially assemble into a parallel configuration, both parallel and antiparallel configurations were observed.

    View details for DOI 10.1529/biophysj.104.048637

    View details for Web of Science ID 000224732500057

    View details for PubMedID 15347585

  • tRNA selection and kinetic proofreading in translation NATURE STRUCTURAL & MOLECULAR BIOLOGY Blanchard, S. C., Gonzalez, R. L., Kim, H. D., Chu, S., Puglisi, J. D. 2004; 11 (10): 1008-1014

    Abstract

    Using single-molecule methods we observed the stepwise movement of aminoacyl-tRNA (aa-tRNA) into the ribosome during selection and kinetic proofreading using single-molecule fluorescence resonance energy transfer (smFRET). Intermediate states in the pathway of tRNA delivery were observed using antibiotics and nonhydrolyzable GTP analogs. We identified three unambiguous FRET states corresponding to initial codon recognition, GTPase-activated and fully accommodated states. The antibiotic tetracycline blocks progression of aa-tRNA from the initial codon recognition state, whereas cleavage of the sarcin-ricin loop impedes progression from the GTPase-activated state. Our data support a model in which ribosomal recognition of correct codon-anticodon pairs drives rotational movement of the incoming complex of EF-Tu-GTP-aa-tRNA toward peptidyl-tRNA during selection on the ribosome. We propose a mechanistic model of initial selection and proofreading.

    View details for DOI 10.1038/nsmb831

    View details for Web of Science ID 000224124200023

    View details for PubMedID 15448679

  • tRNA dynamics on the ribosome during translation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Blanchard, S. C., Kim, H. D., Gonzalez, R. L., Puglisi, J. D., Chu, S. 2004; 101 (35): 12893-12898

    Abstract

    Using single-molecule fluorescence spectroscopy, time-resolved conformational changes between fluorescently labeled tRNA have been characterized within surface-immobilized ribosomes proceeding through a complete cycle of translation elongation. Fluorescence resonance energy transfer was used to observe aminoacyl-tRNA (aa-tRNA) stably accommodating into the aminoacyl site (A site) of the ribosome via a multistep, elongation factor-Tu dependent process. Subsequently, tRNA molecules, bound at the peptidyl site and A site, fluctuate between two configurations assigned as classical and hybrid states. The lifetime of classical and hybrid states, measured for complexes carrying aa-tRNA and peptidyl-tRNA at the A site, shows that peptide bond formation decreases the lifetime of the classical-state tRNA configuration by approximately 6-fold. These data suggest that the growing peptide chain plays a role in modulating fluctuations between hybrid and classical states. Single-molecule fluorescence resonance energy transfer was also used to observe aa-tRNA accommodation coupled with elongation factor G-mediated translocation. Dynamic rearrangements in tRNA configuration are also observed subsequent to the translocation reaction. This work underscores the importance of dynamics in ribosome function and demonstrates single-particle enzymology in a system of more than two components.

    View details for DOI 10.1073/pnas.0403884101

    View details for Web of Science ID 000223694700023

    View details for PubMedID 15317937

  • Risk factors for proximal humerus fracture AMERICAN JOURNAL OF EPIDEMIOLOGY Chu, S. P., Kelsey, J. L., Keegan, T. H., Sternfeld, B., Prill, M., Quesenberry, C. P., Sidney, S. 2004; 160 (4): 360-367

    Abstract

    This case-control study of proximal humerus fracture included 448 incident female and male cases and 2,023 controls aged 45 years or older identified in five Northern California Kaiser Permanente Medical Centers in 1996-2001. Data were collected by using an interviewer-administered questionnaire. Some factors related to low bone mass, including number of fractures since age 45 years and low dietary calcium intake, were associated with increased risks of fracture, and factors thought to protect against bone loss, such as menopausal hormone therapy and calcium carbonate tablet use, were associated with reduced risks. Fall-related risk factors included previous falls, diabetes mellitus, and difficulty walking in dim light. Possible fall-related risk factors suggested for the first time in this study were seizure medication use (adjusted odds ratio (OR) = 2.80, 95% confidence interval (CI): 1.45, 5.42), depression (OR = 1.34, 95% CI: 0.98, 1.84), almost always using a hearing aid (OR = 1.92, 95% CI: 1.12, 3.31 vs. never prescribed), and left-handedness (OR = 2.36, 95% CI: 1.51, 3.68 vs. right-handedness). Difficulty with activities of daily living and lack of physical activity tended to be associated with increased risk. Prevention of falls among frail, osteoporotic persons would likely reduce the frequency of proximal humerus fracture.

    View details for DOI 10.1093/aje/kwh224

    View details for Web of Science ID 000223141400008

    View details for PubMedID 15286021

  • Single-molecule studies of SNARE complex assembly reveal parallel and antiparallel configurations PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Weninger, K., Bowen, M. E., Chu, S., Brunger, A. T. 2003; 100 (25): 14800-14805

    Abstract

    Vesicle fusion in eukaryotes is thought to involve the assembly of a highly conserved family of proteins termed soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) into a highly stable parallel four-helix bundle. We have used intermolecular single-molecule fluorescence resonance energy transfer to characterize preassembled neuronal SNARE complexes consisting of syntaxin, synaptobrevin, and synaptosome-associated protein of 25 kDa on deposited lipid bilayers. Surprisingly, we found a mixture of parallel as well as antiparallel configurations involving the SNARE motifs of syntaxin and synaptobrevin as well as those of syntaxin and synaptosome-associated protein of 25 kDa. The subpopulation with the parallel four-helix bundle configuration could be greatly enriched by an additional purification step in the presence of denaturant, indicating that the parallel configuration is the energetically most favorable state. Interconversion between the configurations was not observed. From this observation, we infer the conversion rate to be <1.5 h-1. The existence of antiparallel configurations suggests a regulatory role of chaperones, such as N-ethylmaleimide-sensitive factor, or the membrane environment during SNARE complex assembly in vivo, and it could be a partial explanation for the relatively slow rates of vesicle fusion observed by reconstituted fusion experiments in vitro.

    View details for DOI 10.1073/pnas.2036428100

    View details for Web of Science ID 000187227200037

    View details for PubMedID 14657376

  • Observation of polymer conformation hysteresis in extensional flow SCIENCE Schroeder, C. M., Babcock, H. P., Shaqfeh, E. S., Chu, S. 2003; 301 (5639): 1515-1519

    Abstract

    Highly extensible Escherichia coli DNA molecules in planar extensional flow were visualized in dilute solution by fluorescence microscopy. For a narrow range of flow strengths, the molecules were found in either a coiled or highly extended conformation, depending on the deformation history of the polymer. This conformation hysteresis persists for many polymer relaxation times and is due to conformation-dependent hydrodynamic forces. Polymer conformational free-energy landscapes were calculated from computer simulations and show two free-energy minima for flow strengths near the coil-stretch transition. Hysteresis cycles may directly influence bulk-solution stresses and the development of stress-strain relations for dilute polymer flows.

    View details for Web of Science ID 000185255300044

    View details for PubMedID 12970560

  • Visualization of molecular fluctuations near the critical point of the coil-stretch transition in polymer elongation MACROMOLECULES Babcock, H. P., Teixeira, R. E., Hur, J. S., Shaqfeh, E. S., Chu, S. 2003; 36 (12): 4544-4548

    View details for DOI 10.1021/ma034073p

    View details for Web of Science ID 000183526900041

  • Exploration of the transition state for tertiary structure formation between an RNA helix and a large structured RNA JOURNAL OF MOLECULAR BIOLOGY Bartley, L. E., Zhuang, X. W., Das, R., Chu, S., Herschlag, D. 2003; 328 (5): 1011-1026

    Abstract

    Docking of the P1 duplex into the pre-folded core of the Tetrahymena group I ribozyme exemplifies the formation of tertiary interactions in the context of a complex, structured RNA. We have applied Phi-analysis to P1 docking, which compares the effects of modifications on the rate constant for docking (k(dock)) with the effects on the docking equilibrium (K(dock)). To accomplish this we used a single molecule fluorescence resonance energy transfer assay that allows direct determination of the rate constants for formation of thermodynamically favorable, as well as unfavorable, states. Modification of the eight groups of the P1 duplex that make tertiary interactions with the core and changes in solution conditions decrease K(dock) up to 500-fold, whereas k(dock) changes by

    View details for Web of Science ID 000182767000004

    View details for PubMedID 12729738

  • Biology and polymer physics at the single-molecule level PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY A-MATHEMATICAL PHYSICAL AND ENGINEERING SCIENCES Chu, S. 2003; 361 (1805): 689-698

    Abstract

    The ability to look at individual molecules has given us new insights into molecular processes. Examples of our recent work are given to illustrate how behaviour that may otherwise be hidden from view can be clearly seen in single-molecule experiments.

    View details for DOI 10.1098/rsta.2002.1157

    View details for Web of Science ID 000182399900010

    View details for PubMedID 12871618

  • Sensitive detection of cold cesium molecules formed on Feshbach resonances PHYSICAL REVIEW LETTERS Chin, C., Kerman, A. J., Vuletic, V., Chu, S. 2003; 90 (3)

    Abstract

    We observe the dynamic formation of quasibound Cs2 molecules near Feshbach resonances in a cold sample of atomic cesium. Using an external probe beam, more than 15 weakly coupled molecular states are detected with high sensitivity, whose collisional formation cross sections are as small as sigma=2 x 10(-16) cm(2). By modeling the molecule formation and dissociation processes with rate equations, we conclude that at an atomic density of 10(13) cm(-3) and temperature of 5 microK, more than 5(1)x10(5) Cs2 molecules in a single rovibrational state coexist with 10(8) Cs atoms in our trap.

    View details for DOI 10.1103/PhysRevLett.90.033201

    View details for Web of Science ID 000180579200015

    View details for PubMedID 12570487

  • 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

  • Dynamics and configurational fluctuations of single DNA molecules in linear mixed flows PHYSICAL REVIEW E Hur, J. S., Shaqfeh, E. S., Babcock, H. P., Chu, S. 2002; 66 (1)

    Abstract

    We examine the dynamics of DNA molecules in mixed flows where the ratio of vorticity to strain rate may be slightly above or below unity via Brownian dynamics simulation. We find that the chain dynamics in these flows are dramatically different than those found for simple shear flow. When the strain rate exceeds vorticity, the dynamics are found to be driven by the extra amount of straining. For vorticity-dominated flows, a periodicity in chain extension is observed with considerable chain deformation.

    View details for DOI 10.1103/PhysRevE.66.011915

    View details for Web of Science ID 000177200500061

    View details for PubMedID 12241392

  • Correlating structural dynamics and function in single ribozyme molecules SCIENCE Zhuang, X. W., Kim, H., Pereira, M. J., Babcock, H. P., Walter, N. G., Chu, S. 2002; 296 (5572): 1473-1476

    Abstract

    We have studied the correlation between structural dynamics and function of the hairpin ribozyme. The enzyme-substrate complex exists in either docked (active) or undocked (inactive) conformations. Using single-molecule fluorescence methods, we found complex structural dynamics with four docked states of distinct stabilities and a strong memory effect where each molecule rarely switches between different docked states. We also found substrate cleavage to be rate-limited by a combination of conformational transitions and reversible chemistry equilibrium. The complex structural dynamics quantitatively explain the heterogeneous cleavage kinetics common to many catalytic RNAs. The intimate coupling of structural dynamics and function is likely a general phenomenon for RNA.

    View details for Web of Science ID 000175910300050

    View details for PubMedID 12029135

  • Mg2+-dependent conformational change of RNA studied by fluorescence correlation and FRET on immobilized single molecules PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kim, H. D., Nienhaus, G. U., Ha, T., Orr, J. W., Williamson, J. R., Chu, S. 2002; 99 (7): 4284-4289

    Abstract

    Fluorescence correlation spectroscopy (FCS) of fluorescence resonant energy transfer (FRET) on immobilized individual fluorophores was used to study the Mg2+-facilitated conformational change of an RNA three-helix junction, a structural element that initiates the folding of the 30S ribosomal subunit. Transitions of the RNA junction between open and folded conformations resulted in fluctuations in fluorescence by FRET. Fluorescence fluctuations occurring between two FRET states on the millisecond time scale were found to be dependent on Mg2+ and Na+ concentrations. Correlation functions of the fluctuations were used to determine transition rates between the two conformations as a function of Mg2+ or Na+ concentration. Both the opening and folding rates were found to vary with changing salt conditions. Assuming specific binding of divalent ions to RNA, the Mg2+ dependence of the observed rates cannot be explained by conformational change induced by Mg2+ binding/unbinding, but is consistent with a model in which the intrinsic conformational change of the RNA junction is altered by uptake of Mg2+ ion(s). This version of FCS/FRET on immobilized single molecules is demonstrated to be a powerful technique in the study of conformational dynamics of biomolecules over time scales ranging from microseconds to seconds.

    View details for Web of Science ID 000174856000030

    View details for PubMedID 11929999

  • Cold atoms and quantum control NATURE Chu, S. 2002; 416 (6877): 206-210

    Abstract

    This overview prefaces a collection of Insight review articles on the physics and applications of laser-cooled atoms. I will cast this work into a historical perspective in which laser cooling and trapping is seen as one of several research directions aimed at controlling the internal and external degrees of freedom of atoms and molecules.

    View details for Web of Science ID 000174348100046

    View details for PubMedID 11894103

  • 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

  • Measurement of an electron's electric dipole moment using Cs atoms trapped in optical lattices PHYSICAL REVIEW A Chin, C., Leiber, V., Vuletic, V., Kerman, A. J., Chu, S. 2001; 63 (3)
  • Dynamics of dilute and semidilute DNA solutions in the start-up of shear flow JOURNAL OF RHEOLOGY Hur, J. S., Shaqfeh, E. S., Babcock, H. P., Smith, D. E., Chu, S. 2001; 45 (2): 421-450
  • Fluorescence quenching: A tool for single-molecule protein-folding study PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Zhuang, X. W., Ha, T., Kim, H. D., Centner, T., Labeit, S., Chu, S. 2000; 97 (26): 14241-14244

    Abstract

    By using titin as a model system, we have demonstrated that fluorescence quenching can be used to study protein folding at the single molecule level. The unfolded titin molecules with multiple dye molecules attached are able to fold to the native state. In the native folded state, the fluorescence from dye molecules is quenched due to the close proximity between the dye molecules. Unfolding of the titin leads to a dramatic increase in the fluorescence intensity. Such a change makes the folded and unfolded states of a single titin molecule clearly distinguishable and allows us to measure the folding dynamics of individual titin molecules in real time. We have also shown that fluorescence quenching can signal folding and unfolding of a small protein with only one immunoglobulin domain.

    View details for Web of Science ID 000165993700047

    View details for PubMedID 11121030

  • High resolution Feshbach spectroscopy of cesium PHYSICAL REVIEW LETTERS Chin, C., Vuletic, V., Kerman, A. J., Chu, S. 2000; 85 (13): 2717-2720
  • Relating the microscopic and macroscopic response of a polymeric fluid in a shearing flow PHYSICAL REVIEW LETTERS Babcock, H. P., Smith, D. E., Hur, J. S., Shaqfeh, E. S., Chu, S. 2000; 85 (9): 2018-2021

    Abstract

    The microscopic and macroscopic response of a polymer solution in start-up shear flow was investigated using fluorescence microscopy of single molecules, bulk viscosity measurements, and Brownian dynamics simulations. An overshoot in viscosity was observed upon flow inception and understood via the observed molecular extension and by simulation findings. Increasing the polymer concentration up to six times the overlap concentration ( C(*)) has no effect on the character of the dynamics of individual molecules.

    View details for Web of Science ID 000088965300060

    View details for PubMedID 10970672

  • A single-molecule study of RNA catalysis and folding SCIENCE Zhuang, X. W., Bartley, L. E., Babcock, H. P., Russell, R., Ha, T. J., Herschlag, D., Chu, S. 2000; 288 (5473): 2048-?

    Abstract

    Using fluorescence microscopy, we studied the catalysis by and folding of individual Tetrahymena thermophila ribozyme molecules. The dye-labeled and surface-immobilized ribozymes used were shown to be functionally indistinguishable from the unmodified free ribozyme in solution. A reversible local folding step in which a duplex docks and undocks from the ribozyme core was observed directly in single-molecule time trajectories, allowing the determination of the rate constants and characterization of the transition state. A rarely populated docked state, not measurable by ensemble methods, was observed. In the overall folding process, intermediate folding states and multiple folding pathways were observed. In addition to observing previously established folding pathways, a pathway with an observed folding rate constant of 1 per second was discovered. These results establish single-molecule fluorescence as a powerful tool for examining RNA folding.

    View details for Web of Science ID 000087687000052

    View details for PubMedID 10856219

  • Laser cooling of atoms, ions, or molecules by coherent scattering PHYSICAL REVIEW LETTERS Vuletic, V., Chu, S. 2000; 84 (17): 3787-3790
  • Beyond optical molasses: 3D Raman sideband cooling of atomic cesium to high phase-space density PHYSICAL REVIEW LETTERS Kerman, A. J., Vuletic, V., Chin, C., Chu, S. 2000; 84 (3): 439-442
  • High resolution feshbach spectroscopy of cesium Physical review letters Chin, C., Vuletic, V., Kerman, A. J., Chu, S. 2000; 85 (13): 2717-20

    Abstract

    We measure high-resolution Feshbach resonance spectra for ultracold cesium atoms colliding in different hyperfine and magnetic sublevels. More than 25 resonances are observed for magnetic fields up to 230 G and their positions are measured with an accuracy down to 0.03 G. From these spectra several ground-state molecular interaction parameters can be extracted with sufficient accuracy to permit for the first time an unambiguous and accurate determination of cesium's ultracold collision properties [P. J. Leo, C. J. Williams, and P. S. Julienne, following Letter, Phys. Rev. Lett. 85, 2721 (2000)].

    View details for PubMedID 10991216

  • Laser cooling of atoms, ions, or molecules by coherent scattering Physical review letters Vuletic, V., Chu, S. 2000; 84 (17): 3787-90

    Abstract

    We point out a laser cooling method for atoms, molecules, or ions at low saturation and large detuning from the particles' resonances. The moving particle modifies the field inside a cavity with a time delay characteristic of the cavity linewidth, while the field acts on the particle via the light shift. The dissipative mechanism can be interpreted as Doppler cooling based on preferential scattering rather than preferential absorption. It depends on particle properties only through the coherent scattering rate, opening new possibilities for optically cooling molecules or interacting atoms.

    View details for PubMedID 11019206

  • Beyond optical molasses: 3D raman sideband cooling of atomic cesium to high phase-space density Physical review letters Kerman, A. J., Vuletic, V., Chin, C., Chu, S. 2000; 84 (3): 439-42

    Abstract

    We demonstrate a simple, general purpose method to cool neutral atoms. A sample containing 3x10(8) cesium atoms prepared in a magneto-optical trap is cooled and simultaneously spin polarized in 10 ms at a density of 1.1x10(11) cm (-3) to a phase space density nlambda(3)(dB) = 1/500, which is almost 3 orders of magnitude higher than attainable in free space with optical molasses. The technique is based on 3D degenerate Raman sideband cooling in optical lattices and remains efficient even at densities where the mean lattice site occupation is close to unity.

    View details for PubMedID 11015933

  • Ligand-induced conformational changes observed in single RNA molecules PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ha, T., Zhuang, X. W., Kim, H. D., Orr, J. W., Williamson, J. R., Chu, S. 1999; 96 (16): 9077-9082

    Abstract

    We present the first demonstration that fluorescence resonance energy transfer can be used to track the motion of a single molecule undergoing conformational changes. As a model system, the conformational changes of individual three-helix junction RNA molecules induced by the binding of ribosomal protein S15 or Mg(2+) ions were studied by changes in single-molecule fluorescence. The transition from an open to a folded configuration was monitored by the change of fluorescence resonance energy transfer between two different dye molecules attached to the ends of two helices in the RNA junction. Averaged behavior of RNA molecules closely resembles that of unlabeled molecules in solution determined by other bulk assays, proving that this approach is viable and suggesting new opportunities for studying protein-nucleic acids interactions. Surprisingly, we observed an anomalously broad distribution of RNA conformations at intermediate ion concentrations that may be attributed to foldability differences among RNA molecules. In addition, an experimental scheme was developed where the real-time response of single molecules can be followed under changing environments. As a demonstration, we repeatedly changed Mg(2+) concentration in the buffer while monitoring single RNA molecules and showed that individual RNA molecules can measure the instantaneous Mg(2+) concentration with 20-ms time resolution, making it the world's smallest Mg(2+) meter.

    View details for Web of Science ID 000081835500056

    View details for PubMedID 10430898

  • Suppression of atomic radiative collisions by tuning the ground state scattering length PHYSICAL REVIEW LETTERS Vuletic, V., Chin, C., Kerman, A. J., Chu, S. 1999; 83 (5): 943-946
  • Single-polymer dynamics in steady shear flow SCIENCE Smith, D. E., Babcock, H. P., Chu, S. 1999; 283 (5408): 1724-1727

    Abstract

    The conformational dynamics of individual, flexible polymers in steady shear flow were directly observed by the use of video fluorescence microscopy. The probability distribution for the molecular extension was determined as a function of shear rate, gamma;, for two different polymer relaxation times, tau. In contrast to the behavior in pure elongational flow, the average polymer extension in shear flow does not display a sharp coil-stretch transition. Large, aperiodic temporal fluctuations were observed, consistent with end-over-end tumbling of the molecule. The rate of these fluctuations (relative to the relaxation rate) increased as the Weissenberg number, gamma;tau, was increased.

    View details for Web of Science ID 000079102800048

    View details for PubMedID 10073935

  • Brownian dynamics simulations of a DNA molecule in an extensional flow field JOURNAL OF RHEOLOGY Larson, R. G., Hu, H., Smith, D. E., Chu, S. 1999; 43 (2): 267-304
  • Observation of low-field Feshbach resonances in collisions of cesium atoms PHYSICAL REVIEW LETTERS Vuletic, V., Kerman, A. J., Chin, C., Chu, S. 1999; 82 (7): 1406-1409
  • Degenerate Raman sideband cooling of trapped cesium atoms at very high atomic densities PHYSICAL REVIEW LETTERS Vuletic, V., Chin, C., Kerman, A. J., Chu, S. 1998; 81 (26): 5768-5771
  • Response of flexible polymers to a sudden elongational flow Science (New York, N.Y.) Smith, D. E., Chu, S. 1998; 281 (5381): 1335-40

    Abstract

    Individual polymers at thermal equilibrium were exposed to an elongational flow producing a high strain rate, and their dynamics were recorded with video fluorescence microscopy. The flow was turned on suddenly so that the entire evolution of molecular conformation could be observed without initial perturbations. The rate of stretching of individual molecules is highly variable and depends on the molecular conformation that develops during stretching. This variability is due to a dependence of the dynamics on the initial, random equilibrium conformation of the polymer coil. The increasing appearance at high strain rates of slowly unraveling hairpin folds is an example of nonergodic dynamics, which can occur when a statistical mechanical system is subjected to nonadiabatic, or "sudden," external forces.

    View details for PubMedID 9721095

  • The dynamics of partially extended single molecules of DNA NATURE Quake, S. R., Babcock, H., Chu, S. 1997; 388 (6638): 151-154

    Abstract

    The behaviour of an isolated polymer floating in a solvent forms the basis of our understanding of polymer dynamics. Classical theories describe the motion of a polymer with linear equations of motion, which yield a set of 'normal modes', analogous to the fundamental frequency and the harmonics of a vibrating violin string. But hydrodynamic interactions make polymer dynamics inherently nonlinear, and the linearizing approximations required for the normal-mode picture have therefore been questioned. Here we test the normal-mode theory by measuring the fluctuations of single molecules of DNA held in a partially extended state with optical tweezers. We find that the motion of the DNA can be described by linearly independent normal modes, and we have experimentally determined the eigenstates of the system. Furthermore, we show that the spectrum of relaxation times obeys a power law.

    View details for Web of Science ID A1997XK10900043

    View details for PubMedID 9217154

  • Single polymer dynamics in an elongational flow SCIENCE Perkins, T. T., Smith, D. E., Chu, S. 1997; 276 (5321): 2016-2021

    Abstract

    The stretching of individual polymers in a spatially homogeneous velocity gradient was observed through use of fluorescently labeled DNA molecules. The probability distribution of molecular extension was determined as a function of time and strain rate. Although some molecules reached steady state, the average extension did not, even after a approximately 300-fold distortion of the underlying fluid element. At the highest strain rates, distinct conformational shapes with differing dynamics were observed. There was considerable variation in the onset of stretching, and chains with a dumbbell shape stretched more rapidly than folded ones. As the strain rate was increased, chains did not deform with the fluid element. The steady-state extension can be described by a model consisting of two beads connected by a spring representing the entropic elasticity of a worm-like chain, but the average dynamics cannot.

    View details for Web of Science ID A1997XG74800057

    View details for PubMedID 9197259

  • Bioreactive self-assembled monolayers on hydrogen-passivated Si(111) as a new class of atomically flat substrates for biological scanning probe microscopy JOURNAL OF STRUCTURAL BIOLOGY Wagner, P., Nock, S., Spudich, J. A., VOLKMUTH, W. D., Chu, S., Cicero, R. L., Wade, C. P., Linford, M. R., Chidsey, C. E. 1997; 119 (2): 189-201

    Abstract

    This is the first report of bioreactive self-assembled monolayers, covalently bound to atomically flat silicon surfaces and capable of binding biomolecules for investigation by scanning probe microscopy and other surface-related assays and sensing devices. These monolayers are stable under a wide range of conditions and allow tailor-made functionalization for many purposes. We describe the substrate preparation and present an STM and SFM characterization, partly performed with multiwalled carbon nanotubes as tapping-mode supertips. Furthermore, we present two strategies of introducing in situ reactive headgroup functionalities. One method entails a free radical chlorosulfonation process with subsequent sulfonamide formation. A second method employs singlet carbenemediated hydrogen-carbon insertion of a heterobifunctional, amino-reactive trifluoromethyl-diazirinyl crosslinker. We believe that this new substrate is advantageous to others, because it (i) is atomically flat over large areas and can be prepared in a few hours with standard equipment, (ii) is stable under most conditions, (iii) can be modified to adjust a certain degree of reactivity and hydrophobicity, which allows physical adsorption or covalent crosslinking of the biological specimen, (iv) builds the bridge between semiconductor microfabrication and organic/biological molecular systems, and (v) is accessible to nanopatterning and applications requiring conductive substrates.

    View details for Web of Science ID A1997XN38200014

    View details for PubMedID 9245759

  • Raman cooling of atoms in an optical dipole trap PHYSICAL REVIEW LETTERS Lee, H. J., Adams, C. S., Kasevich, M., Chu, S. 1996; 76 (15): 2658-2661
  • Quantitative measurements of force and displacement using an optical trap BIOPHYSICAL JOURNAL Simmons, R. M., Finer, J. T., Chu, S., Spudich, J. A. 1996; 70 (4): 1813-1822

    Abstract

    We combined a single-beam gradient optical trap with a high-resolution photodiode position detector to show that an optical trap can be used to make quantitative measurements of nanometer displacements and piconewton forces with millisecond resolution. When an external force is applied to a micron-sized bead held by an optical trap, the bead is displaced from the center of the trap by an amount proportional to the applied force. When the applied force is changed rapidly, the rise time of the displacement is on the millisecond time scale, and thus a trapped bead can be used as a force transducer. The performance can be enhanced by a feedback circuit so that the position of the trap moves by means of acousto-optic modulators to exert a force equal and opposite to the external force applied to the bead. In this case the position of the trap can be used to measure the applied force. We consider parameters of the trapped bead such as stiffness and response time as a function of bead diameter and laser beam power and compare the results with recent ray-optic calculations.

    View details for Web of Science ID A1996UB81800027

    View details for PubMedID 8785341

  • Dynamical scaling of DNA diffusion coefficients MACROMOLECULES Smith, D. E., Perkins, T. T., Chu, S. 1996; 29 (4): 1372-1373
  • STRETCHING OF A SINGLE TETHERED POLYMER IN A UNIFORM-FLOW SCIENCE Perkins, T. T., Smith, D. E., Larson, R. G., Chu, S. 1995; 268 (5207): 83-87

    Abstract

    The stretching of single, tethered DNA molecules by a flow was directly visualized with fluorescence microscopy. Molecules ranging in length (L) from 22 to 84 micrometers were held stationary against the flow by the optical trapping of a latex microsphere attached to one end. The fractional extension x/L is a universal function of eta vL 0.54 +/- 0.05, where eta and v are the viscosity and velocity of the flow, respectively. This relation shows that the DNA is not "free-draining" (that is, hydrodynamic coupling within the chain is not negligible) even near full extension (approximately 80 percent). This function has the same form over a long range as the fractional extension versus force applied at the ends of a worm-like chain. For small deformations (< 30 percent of full extension), the extension increases with velocity as x approximately v0.70 +/- 0.08. The relative size of fluctuations in extension decreases as sigma x/x approximately equal to 0.42 exp (-4.9 x/L). Video images of the fluctuating chain have a cone-like envelope and show a sharp increase in intensity at the free end.

    View details for Web of Science ID A1995QR45400029

    View details for PubMedID 7701345

  • RAMAN COOLING OF ATOMS IN 2-DIMENSIONS AND 3-DIMENSIONS PHYSICAL REVIEW LETTERS Davidson, N., Lee, H. J., Kasevich, M., Chu, S. 1994; 72 (20): 3158-3161
  • RELAXATION OF A SINGLE DNA MOLECULE OBSERVED BY OPTICAL MICROSCOPY SCIENCE Perkins, T. T., Quake, S. R., Smith, D. E., Chu, S. 1994; 264 (5160): 822-826

    Abstract

    Single molecules of DNA, visualized in video fluorescence microscopy, were stretched to full extension in a flow, and their relaxation was measured when the flow stopped. The molecules, attached by one end to a 1-micrometer bead, were manipulated in an aqueous solution with optical tweezers. Inverse Laplace transformations of the relaxation data yielded spectra of decaying exponentials with distinct peaks, and the longest time component (tau) increased with length (L) as tau approximately L 1.68 +/- 0.10. A rescaling analysis showed that most of the relaxation curves had a universal shape and their characteristic times (lambda t) increased as lambda t approximately L 1.65 +/- 0.13. These results are in qualitative agreement with the theoretical prediction of dynamical scaling.

    View details for Web of Science ID A1994NJ94900026

    View details for PubMedID 8171336

  • DIRECT OBSERVATION OF TUBE-LIKE MOTION OF A SINGLE POLYMER-CHAIN SCIENCE Perkins, T. T., Smith, D. E., Chu, S. 1994; 264 (5160): 819-822

    Abstract

    Tube-like motion of a single, fluorescently labeled molecule of DNA in an entangled solution of unlabeled lambda-phage DNA molecules was observed by fluorescence microscopy. One end of a 16- to 100-micrometer-long DNA was attached to a 1-micrometer bead and moved with optical tweezers. The molecule was stretched into various conformations having bends, kinks, and loops. As the polymer relaxed, it closely followed a path defined by its initial contour. The relaxation time of the disturbance caused by the bead was roughly 1 second, whereas tube-like motion in small loops persisted for longer than 2 minutes. Tube deformation, constraint release, and excess chain segment diffusion were also observed. These observations provide direct evidence for several key assumptions in the reptation model developed by de Gennes, Edwards, and Doi.

    View details for Web of Science ID A1994NJ94900025

    View details for PubMedID 8171335

  • LASER-COOLED CS FREQUENCY STANDARD AND A MEASUREMENT OF THE FREQUENCY-SHIFT DUE TO ULTRACOLD COLLISIONS PHYSICAL REVIEW LETTERS Gibble, K., Chu, S. 1993; 70 (12): 1771-1774
  • IN-VITRO METHODS FOR MEASURING FORCE AND VELOCITY OF THE ACTIN-MYOSIN INTERACTION USING PURIFIED PROTEINS METHODS IN CELL BIOLOGY, VOL 39 Warrick, H. M., Simmons, R. M., Finer, J. T., Uyeda, T. Q., Chu, S., Spudich, J. A. 1993; 39: 1-21

    View details for Web of Science ID A1993BZ58U00001

    View details for PubMedID 8246790

  • LASER COOLING BELOW A PHOTON RECOIL WITH 3-LEVEL ATOMS PHYSICAL REVIEW LETTERS Kasevich, M., Chu, S. 1992; 69 (12): 1741-1744
  • MEASUREMENT OF THE GRAVITATIONAL ACCELERATION OF AN ATOM WITH A LIGHT-PULSE ATOM INTERFEROMETER APPLIED PHYSICS B-PHOTOPHYSICS AND LASER CHEMISTRY Kasevich, M., Chu, S. 1992; 54 (5): 321-332
  • FUTURE SLOW-ATOM FREQUENCY STANDARDS METROLOGIA Gibble, K., Chu, S. 1992; 29 (2): 201-212
  • IMPROVED MAGNETOOPTIC TRAPPING IN A VAPOR CELL OPTICS LETTERS GIBBLE, K. E., Kasapi, S., Chu, S. 1992; 17 (7): 526-528

    Abstract

    We have captured 3.6 x 10(10) cesium atoms in a magneto-optic trap loaded from a vapor cell. The 300-fold increase in the number of trapped atoms compared with that of previous research was accomplished by using larger laser intensities and 4-cm-diameter laser beams. The loading time constant was as short as 0.2 s.

    View details for Web of Science ID A1992HK74400022

    View details for PubMedID 19794547

  • LASER TRAPPING OF NEUTRAL PARTICLES SCIENTIFIC AMERICAN Chu, S. 1992; 266 (2): 70-76
  • THEORETICAL-ANALYSIS OF VELOCITY-SELECTIVE RAMAN TRANSITIONS PHYSICAL REVIEW A Moler, K., Weiss, D. S., Kasevich, M., Chu, S. 1992; 45 (1): 342-348
  • ATOMIC INTERFEROMETRY USING STIMULATED RAMAN TRANSITIONS PHYSICAL REVIEW LETTERS Kasevich, M., Chu, S. 1991; 67 (2): 181-184
  • ATOMIC VELOCITY SELECTION USING STIMULATED RAMAN TRANSITIONS PHYSICAL REVIEW LETTERS Kasevich, M., Weiss, D. S., Riis, E., Moler, K., Kasapi, S., Chu, S. 1991; 66 (18): 2297-2300
  • NORMAL-INCIDENCE REFLECTION OF SLOW ATOMS FROM AN OPTICAL EVANESCENT WAVE OPTICS LETTERS Kasevich, M. A., Weiss, D. S., Chu, S. 1990; 15 (11): 607-609

    View details for Web of Science ID A1990DG12400005

    View details for PubMedID 19768022

  • OPTICAL MOLASSES AND MULTILEVEL ATOMS - THEORY JOURNAL OF THE OPTICAL SOCIETY OF AMERICA B-OPTICAL PHYSICS UNGAR, P. J., Weiss, D. S., Riis, E., Chu, S. 1989; 6 (11): 2058-2071
  • OPTICAL MOLASSES AND MULTILEVEL ATOMS - EXPERIMENT JOURNAL OF THE OPTICAL SOCIETY OF AMERICA B-OPTICAL PHYSICS Weiss, D. S., Riis, E., Shevy, Y., UNGAR, P. J., Chu, S. 1989; 6 (11): 2072-2083
  • RF SPECTROSCOPY IN AN ATOMIC FOUNTAIN PHYSICAL REVIEW LETTERS Kasevich, M. A., Riis, E., Chu, S., DeVoe, R. G. 1989; 63 (6): 612-616
  • BIMODAL SPEED DISTRIBUTIONS IN LASER-COOLED ATOMS PHYSICAL REVIEW LETTERS Shevy, Y., Weiss, D. S., UNGAR, P. J., Chu, S. 1989; 62 (10): 1118-1121

Conference Proceedings


  • Towards Structural Biology with Single Molecules Brunger, A., Strop, P., Vrljic, M., Chu, S., Weninger, K. FEDERATION AMER SOC EXP BIOL. 2011
  • Single molecule studies of SNARE-dependent fusion Brunger, A., Chu, S., Bowen, M., Weninger, K., Vrljic, M. CELL PRESS. 2007: 375A-375A
  • PRECISION-MEASUREMENTS WITH COLD ATOMS Weiss, D. S., Gibble, K., Young, B., Peters, A., Chu, S. AIP PRESS. 1994: 23-28
  • PRECISION ATOM INTERFEROMETRY AND AN IMPROVED MEASUREMENT OF THE 13S1-23S1 TRANSITION IN POSITRONIUM Chu, S. ELSEVIER SCIENCE PUBL B V. 1994: 317-355
  • MEASUREMENT OF THE ACCELERATION DUE TO GRAVITY WITH AN ATOMIC INTERFEROMETER Kasevich, M., Chu, S. WORLD SCIENTIFIC PUBL CO PTE LTD. 1992: 47-54

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