Bio

Bio

Molecular motors lie at the heart of biological processes from DNA replication to vesicle transport. My laboratory seeks to understand the physical mechanisms by which these nanoscale machines convert chemical energy into mechanical work. We use single molecule tracking and manipulation techniques to observe and perturb substeps in the mechanochemical cycles of individual motors. Protein engineering helps us to explore relationships between molecular structures and mechanical functions. Broad topics of current interest include torque generation by DNA-associated ATPases and mechanical adaptations of unconventional myosins.

Academic Appointments

Administrative Appointments

  • Associate Chair of Graduate Admissions, Stanford Bioengineering (2018 - Present)

Honors & Awards

  • Predoctoral Fellowship, Howard Hughes Medical Institute (1999)
  • Harold M. Weintraub Award, FHCRC (2004)
  • Alan Bearden Award, UC, Berkeley (2004)
  • Postdoctoral Fellowship, Helen Hay Whitney Foundation (2005)
  • Director's New Innovator Award, NIH (2008)
  • Pew Scholars Award, Pew Charitable Trusts (2009)

Boards, Advisory Committees, Professional Organizations

  • Council Member, Biophysical Society (2017 - 2020)

Professional Education

  • B.Sc., University of Washington, Biochemistry (1998)
  • Ph.D., UC, Berkeley, Molecular and Cell Biology (2003)

Contact

Links

Research & Scholarship

Current Research and Scholarly Interests

Molecular motors lie at the heart of biological processes from DNA replication to vesicle transport. My laboratory seeks to understand the physical mechanisms by which these nanoscale machines convert chemical energy into mechanical work. We use single molecule tracking and manipulation techniques to observe and perturb substeps in the mechanochemical cycles of individual motors. Protein engineering helps us to explore relationships between molecular structures and mechanical functions. Broad topics of current interest include torque generation by DNA-associated ATPases and mechanical adaptations of unconventional myosins.

Teaching

2020-21 Courses

Stanford Advisees

Graduate and Fellowship Programs

Publications

All Publications

  • Optical control of fast and processive engineered myosins in vitro and in living cells. Nature chemical biology Ruijgrok, P. V., Ghosh, R. P., Zemsky, S., Nakamura, M., Gong, R., Ning, L., Chen, R., Vachharajani, V. T., Chu, A. E., Anand, N., Eguchi, R. R., Huang, P. S., Lin, M. Z., Alushin, G. M., Liphardt, J. T., Bryant, Z. 2021

    Abstract

    Precision tools for spatiotemporal control of cytoskeletal motor function are needed to dissect fundamental biological processes ranging from intracellular transport to cell migration and division. Direct optical control of motor speed and direction is one promising approach, but it remains a challenge to engineer controllable motors with desirable properties such as the speed and processivity required for transport applications in living cells. Here, we develop engineered myosin motors that combine large optical modulation depths with high velocities, and create processive myosin motors with optically controllable directionality. We characterize the performance of the motors using in vitro motility assays, single-molecule tracking and live-cell imaging. Bidirectional processive motors move efficiently toward the tips of cellular protrusions in the presence of blue light, and can transport molecular cargo in cells. Robust gearshifting myosins will further enable programmable transport in contexts ranging from in vitro active matter reconstitutions to microfabricated systems that harness molecular propulsion.

    View details for DOI 10.1038/s41589-021-00740-7

    View details for PubMedID 33603247

  • Cas9 interrogates DNA in discrete steps modulated by mismatches and supercoiling. Proceedings of the National Academy of Sciences of the United States of America Ivanov, I. E., Wright, A. V., Cofsky, J. C., Aris, K. D., Doudna, J. A., Bryant, Z. 2020

    Abstract

    The CRISPR-Cas9 nuclease has been widely repurposed as a molecular and cell biology tool for its ability to programmably target and cleave DNA. Cas9 recognizes its target site by unwinding the DNA double helix and hybridizing a 20-nucleotide section of its associated guide RNA to one DNA strand, forming an R-loop structure. A dynamic and mechanical description of R-loop formation is needed to understand the biophysics of target searching and develop rational approaches for mitigating off-target activity while accounting for the influence of torsional strain in the genome. Here we investigate the dynamics of Cas9 R-loop formation and collapse using rotor bead tracking (RBT), a single-molecule technique that can simultaneously monitor DNA unwinding with base-pair resolution and binding of fluorescently labeled macromolecules in real time. By measuring changes in torque upon unwinding of the double helix, we find that R-loop formation and collapse proceed via a transient discrete intermediate, consistent with DNA:RNA hybridization within an initial seed region. Using systematic measurements of target and off-target sequences under controlled mechanical perturbations, we characterize position-dependent effects of sequence mismatches and show how DNA supercoiling modulates the energy landscape of R-loop formation and dictates access to states competent for stable binding and cleavage. Consistent with this energy landscape model, in bulk experiments we observe promiscuous cleavage under physiological negative supercoiling. The detailed description of DNA interrogation presented here suggests strategies for improving the specificity and kinetics of Cas9 as a genome engineering tool and may inspire expanded applications that exploit sensitivity to DNA supercoiling.

    View details for DOI 10.1073/pnas.1913445117

    View details for PubMedID 32123105

  • Controllable molecular motors engineered from myosin and RNA. Nature nanotechnology Omabegho, T., Gurel, P. S., Cheng, C. Y., Kim, L. Y., Ruijgrok, P. V., Das, R., Alushin, G. M., Bryant, Z. 2017

    Abstract

    Engineering biomolecular motors can provide direct tests of structure-function relationships and customized components for controlling molecular transport in artificial systems 1 or in living cells 2 . Previously, synthetic nucleic acid motors 3-5 and modified natural protein motors 6-10 have been developed in separate complementary strategies to achieve tunable and controllable motor function. Integrating protein and nucleic-acid components to form engineered nucleoprotein motors may enable additional sophisticated functionalities. However, this potential has only begun to be explored in pioneering work harnessing DNA scaffolds to dictate the spacing, number and composition of tethered protein motors 11-15 . Here, we describe myosin motors that incorporate RNA lever arms, forming hybrid assemblies in which conformational changes in the protein motor domain are amplified and redirected by nucleic acid structures. The RNA lever arm geometry determines the speed and direction of motor transport and can be dynamically controlled using programmed transitions in the lever arm structure 7,9 . We have characterized the hybrid motors using in vitro motility assays, single-molecule tracking, cryo-electron microscopy and structural probing 16 . Our designs include nucleoprotein motors that reversibly change direction in response to oligonucleotides that drive strand-displacement 17 reactions. In multimeric assemblies, the controllable motors walk processively along actin filaments at speeds of 10-20?nm?s-1. Finally, to illustrate the potential for multiplexed addressable control, we demonstrate sequence-specific responses of RNA variants to oligonucleotide signals.

    View details for PubMedID 29109539

  • Multimodal Measurements of Single-Molecule Dynamics Using FluoRBT. Biophysical journal Ivanov, I. E., Lebel, P., Oberstrass, F. C., Starr, C. H., Parente, A. C., Ierokomos, A., Bryant, Z. 2017

    Abstract

    Single-molecule methods provide direct measurements of macromolecular dynamics, but are limited by the number of degrees of freedom that can be followed at one time. High-resolution rotor bead tracking (RBT) measures DNA torque, twist, and extension, and can be used to characterize the structural dynamics of DNA and diverse nucleoprotein complexes. Here, we extend RBT to enable simultaneous monitoring of additional degrees of freedom. Fluorescence-RBT (FluoRBT) combines magnetic tweezers, infrared evanescent scattering, and single-molecule FRET imaging, providing real-time multiparameter measurements of complex molecular processes. We demonstrate the capabilities of FluoRBT by conducting simultaneous measurements of extension and FRET during opening and closing of a DNA hairpin under tension, and by observing simultaneous changes in FRET and torque during a transition between right-handed B-form and left-handed Z-form DNA under controlled supercoiling. We discover unanticipated continuous changes in FRET with applied torque, and also show how FluoRBT can facilitate high-resolution FRET measurements of molecular states, by using a mechanical signal as an independent temporal reference for aligning and averaging noisy fluorescence data. By combining mechanical measurements of global DNA deformations with FRET measurements of local conformational changes, FluoRBT will enable multidimensional investigations of systems ranging from DNA structures to large macromolecular machines.

    View details for PubMedID 29248150

  • Gold rotor bead tracking for high-speed measurements of DNA twist, torque and extension. Nature methods Lebel, P., Basu, A., Oberstrass, F. C., Tretter, E. M., Bryant, Z. 2014; 11 (4): 456-462

    Abstract

    Single-molecule measurements of DNA twist and extension have been used to reveal physical properties of the double helix and to characterize structural dynamics and mechanochemistry in nucleoprotein complexes. However, the spatiotemporal resolution of twist measurements has been limited by the use of angular probes with high rotational drag, which prevents detection of short-lived intermediates or small angular steps. We introduce gold rotor bead tracking (AuRBT), which yields >100× improvement in time resolution over previous techniques. AuRBT employs gold nanoparticles as bright low-drag rotational and extensional probes, which are monitored by instrumentation that combines magnetic tweezers with objective-side evanescent darkfield microscopy. Our analysis of high-speed structural dynamics of DNA gyrase using AuRBT revealed an unanticipated transient intermediate. AuRBT also enables direct measurements of DNA torque with >50× shorter integration times than previous techniques; we demonstrated high-resolution torque spectroscopy by mapping the conformational landscape of a Z-forming DNA sequence.

    View details for DOI 10.1038/nmeth.2854

    View details for PubMedID 24562422

  • Spatiotemporal control of liquid crystal structure and dynamics through activity patterning. Nature materials Zhang, R., Redford, S. A., Ruijgrok, P. V., Kumar, N., Mozaffari, A., Zemsky, S., Dinner, A. R., Vitelli, V., Bryant, Z., Gardel, M. L., de Pablo, J. J. 2021

    Abstract

    Active materials are capable of converting free energy into mechanical work to produce autonomous motion, and exhibit striking collective dynamics that biology relies on for essential functions. Controlling those dynamics and transport in synthetic systems has been particularly challenging. Here, we introduce the concept of spatially structured activity as a means of controlling and manipulating transport in active nematic liquid crystals consisting of actin filaments and light-sensitive myosin motors. Simulations and experiments are used to demonstrate that topological defects can be generated at will and then constrained to move along specified trajectories by inducing local stresses in an otherwise passive material. These results provide a foundation for the design of autonomous and reconfigurable microfluidic systems where transport is controlled by modulating activity with light.

    View details for DOI 10.1038/s41563-020-00901-4

    View details for PubMedID 33603187

  • Modulated control of DNA supercoiling balance by the DNA-wrapping domain of bacterial gyrase. Nucleic acids research Hobson, M. J., Bryant, Z., Berger, J. M. 2020

    Abstract

    Negative supercoiling by DNA gyrase is essential for maintaining chromosomal compaction, transcriptional programming, and genetic integrity in bacteria. Questions remain as to how gyrases from different species have evolved profound differences in their kinetics, efficiency, and extent of negative supercoiling. To explore this issue, we analyzed homology-directed mutations in the C-terminal, DNA-wrapping domain of the GyrA subunit of Escherichia coli gyrase (the 'CTD'). The addition or removal of select, conserved basic residues markedly impacts both nucleotide-dependent DNA wrapping and supercoiling by the enzyme. Weakening CTD-DNA interactions slows supercoiling, impairs DNA-dependent ATP hydrolysis, and limits the extent of DNA supercoiling, while simultaneously enhancing decatenation and supercoil relaxation. Conversely, strengthening DNA wrapping does not result in a more extensively supercoiled DNA product, but partially uncouples ATP turnover from strand passage, manifesting in futile cycling. Our findings indicate that the catalytic cycle of E. coli gyrase operates at high thermodynamic efficiency, and that the stability of DNA wrapping by the CTD provides one limit to DNA supercoil introduction, beyond which strand passage competes with ATP-dependent supercoil relaxation. These results highlight a means by which gyrase can evolve distinct homeostatic supercoiling setpoints in a species-specific manner.

    View details for DOI 10.1093/nar/gkz1230

    View details for PubMedID 31950157

  • Introduction: Molecular Motors. Chemical reviews Iino, R., Kinbara, K., Bryant, Z. 2020; 120 (1): 1?4

    View details for DOI 10.1021/acs.chemrev.9b00819

    View details for PubMedID 31910626

  • Multi-parameter measurements of conformational dynamics in nucleic acids and nucleoprotein complexes. Methods (San Diego, Calif.) Ivanov, I. E., Bryant, Z. 2019

    Abstract

    Biological macromolecules undergo dynamic conformational changes. Single-molecule methods can track such structural rearrangements in real time. However, while the structure of large macromolecules may change along many degrees of freedom, single-molecule techniques only monitor a limited number of these axes of motion. Advanced single-molecule methods are being developed to track multiple degrees of freedom in nucleic acids and nucleoprotein complexes at high resolution, to enable better manipulation and control of the system under investigation, and to collect measurements in massively parallel fashion. Combining complementary single-molecule methods within the same assay also provides unique measurement opportunities. Implementations of magnetic and optical tweezers combined with fluorescence and FRET have demonstrated results unattainable by either technique alone. Augmenting other advanced single-molecule methods with fluorescence detection will allow us to better capture the multidimensional dynamics of nucleic acids and nucleoprotein complexes central to biology.

    View details for DOI 10.1016/j.ymeth.2019.06.019

    View details for PubMedID 31228549

  • A Mechanosensitive RhoA Pathway that Protects Epithelia against Acute Tensile Stress. Developmental cell Acharya, B. R., Nestor-Bergmann, A., Liang, X., Gupta, S., Duszyc, K., Gauquelin, E., Gomez, G. A., Budnar, S., Marcq, P., Jensen, O. E., Bryant, Z., Yap, A. S. 2018

    Abstract

    Adherens junctions are tensile structures that couple epithelial cells together. Junctional tension can arise from cell-intrinsic application of contractility or from the cell-extrinsic forces of tissue movement. Here, we report a mechanosensitive signaling pathway that activates RhoA at adherens junctions to preserve epithelial integrity in response to acute tensile stress. We identify Myosin VI as the force sensor, whose association with E-cadherin is enhanced when junctional tension is increased by mechanical monolayer stress. Myosin VI promotes recruitment of the heterotrimeric Galpha12 protein to E-cadherin, where it signals for p114 RhoGEF to activate RhoA. Despite its potential to stimulate junctional actomyosin and further increase contractility, tension-activated RhoA signaling is necessary to preserve epithelial integrity. This is explained by an increase in tensile strength, especially at the multicellular vertices of junctions, that is due to mDia1-mediated actin assembly.

    View details for PubMedID 30318244

  • Dynamic coupling between conformations and nucleotide states in DNA gyrase. Nature chemical biology Basu, A., Hobson, M., Lebel, P., Fernandes, L. E., Tretter, E. M., Berger, J. M., Bryant, Z. 2018; 14 (6): 565?74

    Abstract

    Gyrase is an essential bacterial molecular motor that supercoils DNA using a conformational cycle in which chiral wrapping of >100 base pairs confers directionality on topoisomerization. To understand the mechanism of this nucleoprotein machine, global structural transitions must be mapped onto the nucleotide cycle of ATP binding, hydrolysis and product release. Here we investigate coupling mechanisms using single-molecule tracking of DNA rotation and contraction during Escherichia coli gyrase activity under varying nucleotide conditions. We find that ADP must be exchanged for ATP to drive the rate-limiting remodeling transition that generates the chiral wrap. ATP hydrolysis accelerates subsequent duplex strand passage and is required for resetting the enzyme and recapturing transiently released DNA. Our measurements suggest how gyrase coordinates DNA rearrangements with the dynamics of its ATP-driven protein gate, how the motor minimizes futile cycles of ATP hydrolysis and how gyrase may respond to changing cellular energy levels to link gene expression with metabolism.

    View details for PubMedID 29662209

  • Rotation of endosomes demonstrates coordination of molecular motors during axonal transport. Science advances Kaplan, L., Ierokomos, A., Chowdary, P., Bryant, Z., Cui, B. 2018; 4 (3): e1602170

    Abstract

    Long-distance axonal transport is critical to the maintenance and function of neurons. Robust transport is ensured by the coordinated activities of multiple molecular motors acting in a team. Conventional live-cell imaging techniques used in axonal transport studies detect this activity by visualizing the translational dynamics of a cargo. However, translational measurements are insensitive to torques induced by motor activities. By using gold nanorods and multichannel polarization microscopy, we simultaneously measure the rotational and translational dynamics for thousands of axonally transported endosomes. We find that the rotational dynamics of an endosome provide complementary information regarding molecular motor activities to the conventionally tracked translational dynamics. Rotational dynamics correlate with translational dynamics, particularly in cases of increased rotation after switches between kinesin- and dynein-mediated transport. Furthermore, unambiguous measurement of nanorod angle shows that endosome-contained nanorods align with the orientation of microtubules, suggesting a direct mechanical linkage between the ligand-receptor complex and the microtubule motors.

    View details for PubMedID 29536037

    View details for PubMedCentralID PMC5846296

  • Cryo-EM structures reveal specialization at the myosin VI-actin interface and a mechanism of force sensitivity. eLife Gurel, P. S., Kim, L. Y., Ruijgrok, P. V., Omabegho, T., Bryant, Z., Alushin, G. M. 2017; 6

    Abstract

    Despite extensive scrutiny of the myosin superfamily, the lack of high-resolution structures of actin-bound states has prevented a complete description of its mechanochemical cycle and limited insight into how sequence and structural diversification of the motor domain gives rise to specialized functional properties. Here we present cryo-EM structures of the unique minus-end directed myosin VI motor domain in rigor (4.6 Ĺ) and Mg-ADP (5.5 Ĺ) states bound to F-actin. Comparison to the myosin IIC-F-actin rigor complex reveals an almost complete lack of conservation of residues at the actin-myosin interface despite preservation of the primary sequence regions composing it, suggesting an evolutionary path for motor specialization. Additionally, analysis of the transition from ADP to rigor provides a structural rationale for force sensitivity in this step of the mechanochemical cycle. Finally, we observe reciprocal rearrangements in actin and myosin accompanying the transition between these states, supporting a role for actin structural plasticity during force generation by myosin VI.

    View details for PubMedID 29199952

  • Structural Dynamics and Mechanochemical Coupling in DNA Gyrase JOURNAL OF MOLECULAR BIOLOGY Basul, A., Parentez, A. C., Bryant, Z. 2016; 428 (9): 1833-1845

    Abstract

    Gyrase is a molecular motor that harnesses the free energy of ATP hydrolysis to perform mechanical work on DNA. The enzyme specifically introduces negative supercoiling in a process that must coordinate fuel consumption with DNA cleavage and religation and with numerous conformational changes in both the protein and DNA components of a large nucleoprotein complex. Here we present a current understanding of mechanochemical coupling in this essential molecular machine, with a focus on recent diverse biophysical approaches that have revealed details of molecular architectures, new conformational intermediates, structural transitions modulated by ATP binding, and the influence of mechanics on motor function. Recent single-molecule assays have also illuminated the reciprocal relationships between supercoiling and transcription, an illustration of mechanical interactions between gyrase and other molecular machines at the heart of chromosomal biology.

    View details for DOI 10.1016/j.jmb.2016.03.016

    View details for Web of Science ID 000376695100003

    View details for PubMedID 27016205

    View details for PubMedCentralID PMC5083069

  • Remote control of myosin and kinesin motors using light-activated gearshifting. Nature nanotechnology Nakamura, M., Chen, L., Howes, S. C., Schindler, T. D., Nogales, E., Bryant, Z. 2014; 9 (9): 693-697

    Abstract

    Cytoskeletal motors perform critical force generation and transport functions in eukaryotic cells. Engineered modifications of motor function provide direct tests of protein structure-function relationships and potential tools for controlling cellular processes or for harnessing molecular transport in artificial systems. Here, we report the design and characterization of a panel of cytoskeletal motors that reversibly change gears-speed up, slow down or switch directions-when exposed to blue light. Our genetically encoded structural designs incorporate a photoactive protein domain to enable light-dependent conformational changes in an engineered lever arm. Using in vitro motility assays, we demonstrate robust spatiotemporal control over motor function and characterize the kinetics of the optical gearshifting mechanism. We have used a modular approach to create optical gearshifting motors for both actin-based and microtubule-based transport.

    View details for DOI 10.1038/nnano.2014.147

    View details for PubMedID 25086603

  • Engineering myosins for long-range transport on actin filaments NATURE NANOTECHNOLOGY Schindler, T. D., Chen, L., Lebel, P., Nakamura, M., Bryant, Z. 2014; 9 (1): 33-38

    Abstract

    Cytoskeletal motors act as cargo transporters in cells and may be harnessed for directed transport applications in molecular detection and diagnostic devices. High processivity, the ability to take many steps along a track before dissociating, is often a desirable characteristic because it allows nanoscale motors to transport cargoes over distances on the scale of micrometres, in vivo and in vitro. Natural processive myosins are dimeric and use internal tension to coordinate the detachment cycles of the two heads. Here, we show that processivity can be enhanced in engineered myosins using two non-natural strategies designed to optimize the effectiveness of random, uncoordinated stepping: (1) the formation of three-headed and four-headed myosins and (2) the introduction of flexible elements between heads. We quantify improvements using systematic single-molecule characterization of a panel of engineered motors. To test the modularity of our approach, we design a controllably bidirectional myosin that is robustly processive in both forward and backward directions, and also produce the fastest processive cytoskeletal motor measured so far, reaching a speed of 10 µm s(-1).

    View details for DOI 10.1038/NNANO.2013.229

    View details for Web of Science ID 000329315000011

    View details for PubMedID 24240432

  • Processive ATP-driven substrate disassembly by the N-ethylmaleimide-sensitive factor (NSF) molecular machine. journal of biological chemistry Cipriano, D. J., Jung, J., Vivona, S., Fenn, T. D., Brunger, A. T., Bryant, Z. 2013; 288 (32): 23436-23445

    Abstract

    SNARE proteins promote membrane fusion by forming a 4-stranded parallel helical bundle that brings the membranes into close proximity. Post fusion, the complex is disassembled by an AAA+ ATPase called N-ethylmaleimide sensitive factor (NSF). We present evidence that NSF uses a processive unwinding mechanism to disassemble SNARE proteins. Using a real-time disassembly assay based on fluorescence dequenching, we correlate NSF-driven disassembly rates with the SNARE-activated ATPase activity of NSF. Neuronal SNAREs activate the ATPase rate of NSF by ~26-fold. One SNARE complex takes an average of ~5 seconds to disassemble in a process that consumes ~50 ATP. Investigations of substrate requirements show that NSF is capable of disassembling a truncated SNARE substrate consisting of only the core SNARE domain, but not an unrelated four-stranded coiled coil. NSF can also disassemble an engineered double-length SNARE complex, suggesting a processive unwinding mechanism. We further investigated processivity using single turnover experiments, which show that SNAREs can be unwound in a single encounter with NSF. We propose a processive helicase-like mechanism for NSF in which ~1 residue is unwound for every hydrolyzed ATP molecule.

    View details for DOI 10.1074/jbc.M113.476705

    View details for PubMedID 23775070

  • Torque Spectroscopy of DNA: Base-Pair Stability, Boundary Effects, Backbending, and Breathing Dynamics. Physical review letters Oberstrass, F. C., Fernandes, L. E., Lebel, P., Bryant, Z. 2013; 110 (17): 178103-?

    View details for PubMedID 23679785

  • Recent developments in single-molecule DNA mechanics CURRENT OPINION IN STRUCTURAL BIOLOGY Bryant, Z., Oberstrass, F. C., Basu, A. 2012; 22 (3): 304-312

    Abstract

    Over the past two decades, measurements on individual stretched and twisted DNA molecules have helped define the basic elastic properties of the double helix and enabled real-time functional assays of DNA-associated molecular machines. Recently, new magnetic tweezers approaches for simultaneously measuring freely fluctuating twist and extension have begun to shed light on the structural dynamics of large nucleoprotein complexes. Related technical advances have facilitated direct measurements of DNA torque, contributing to a better understanding of abrupt structural transitions in mechanically stressed DNA. The new measurements have also been exploited in studies that hint at a developing synergistic relationship between single-molecule manipulation and structural DNA nanotechnology.

    View details for DOI 10.1016/j.sbi.2012.04.007

    View details for Web of Science ID 000306347800008

    View details for PubMedID 22658779

  • ATP binding controls distinct structural transitions of Escherichia coli DNA gyrase in complex with DNA NATURE STRUCTURAL & MOLECULAR BIOLOGY Basu, A., Schoeffler, A. J., Berger, J. M., Bryant, Z. 2012; 19 (5): 538-U105

    Abstract

    DNA gyrase is a molecular motor that harnesses the free energy of ATP hydrolysis to introduce negative supercoils into DNA. A critical step in this reaction is the formation of a chiral DNA wrap. Here we observe gyrase structural dynamics using a single-molecule assay in which gyrase drives the processive, stepwise rotation of a nanosphere attached to the side of a stretched DNA molecule. Analysis of rotational pauses and measurements of DNA contraction reveal multiple ATP-modulated structural transitions. DNA wrapping is coordinated with the ATPase cycle and proceeds by way of an unanticipated structural intermediate that dominates the kinetics of supercoiling. Our findings reveal a conformational landscape of loosely coupled transitions funneling the motor toward productive energy transduction, a feature that may be common to the reaction cycles of other DNA and protein remodeling machines.

    View details for DOI 10.1038/nsmb.2278

    View details for Web of Science ID 000303611200013

    View details for PubMedID 22484318

  • Torque measurements reveal sequence-specific cooperative transitions in supercoiled DNA PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Oberstrass, F. C., Fernandes, L. E., Bryant, Z. 2012; 109 (16): 6106-6111

    Abstract

    B-DNA becomes unstable under superhelical stress and is able to adopt a wide range of alternative conformations including strand-separated DNA and Z-DNA. Localized sequence-dependent structural transitions are important for the regulation of biological processes such as DNA replication and transcription. To directly probe the effect of sequence on structural transitions driven by torque, we have measured the torsional response of a panel of DNA sequences using single molecule assays that employ nanosphere rotational probes to achieve high torque resolution. The responses of Z-forming d(pGpC)(n) sequences match our predictions based on a theoretical treatment of cooperative transitions in helical polymers. "Bubble" templates containing 50-100 bp mismatch regions show cooperative structural transitions similar to B-DNA, although less torque is required to disrupt strand-strand interactions. Our mechanical measurements, including direct characterization of the torsional rigidity of strand-separated DNA, establish a framework for quantitative predictions of the complex torsional response of arbitrary sequences in their biological context.

    View details for DOI 10.1073/pnas.1113532109

    View details for Web of Science ID 000303246100045

    View details for PubMedID 22474350

    View details for PubMedCentralID PMC3341030

  • Engineering controllable bidirectional molecular motors based on myosin NATURE NANOTECHNOLOGY Chen, L., Nakamura, M., Schindler, T. D., Parker, D., Bryant, Z. 2012; 7 (4): 252-256

    Abstract

    Cytoskeletal motors drive the transport of organelles and molecular cargoes within cells and have potential applications in molecular detection and diagnostic devices. Engineering molecular motors with controllable properties will allow selective perturbation of mechanical processes in living cells and provide optimized device components for tasks such as molecular sorting and directed assembly. Biological motors have previously been modified by introducing activation/deactivation switches that respond to metal ions and other signals. Here, we show that myosin motors can be engineered to reversibly change their direction of motion in response to a calcium signal. Building on previous protein engineering studies and guided by a structural model for the redirected power stroke of myosin VI, we have constructed bidirectional myosins through the rigid recombination of structural modules. The performance of the motors was confirmed using gliding filament assays and single fluorophore tracking. Our strategy, in which external signals trigger changes in the geometry and mechanics of myosin lever arms, should make it possible to achieve spatiotemporal control over a range of motor properties including processivity, stride size and branchpoint turning.

    View details for DOI 10.1038/NNANO.2012.19

    View details for Web of Science ID 000302578300012

    View details for PubMedID 22343382

    View details for PubMedCentralID PMC3332125

  • Detailed Tuning of Structure and Intramolecular Communication Are Dispensable for Processive Motion of Myosin VI BIOPHYSICAL JOURNAL Elting, M. W., Bryant, Z., Liao, J., Spudich, J. A. 2011; 100 (2): 430-439

    Abstract

    Dimeric myosin VI moves processively hand-over-hand along actin filaments. We have characterized the mechanism of this processive motion by measuring the impact of structural and chemical perturbations on single-molecule processivity. Processivity is maintained despite major alterations in lever arm structure, including replacement of light chain binding regions and elimination of the medial tail. We present kinetic models that can explain the ATP concentration-dependent processivities of myosin VI constructs containing either native or artificial lever arms. We conclude that detailed tuning of structure and intramolecular communication are dispensable for processive motion, and further show theoretically that one proposed type of nucleotide gating can be detrimental rather than beneficial for myosin processivity.

    View details for DOI 10.1016/j.bpj.2010.11.045

    View details for Web of Science ID 000286543600020

    View details for PubMedID 21244839

    View details for PubMedCentralID PMC3021667

  • Contribution of the myosin VI tail domain to processive stepping and intramolecular tension sensing PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Dunn, A. R., Chuan, P., Bryant, Z., Spudich, J. A. 2010; 107 (17): 7746-7750

    Abstract

    Myosin VI is proposed to act as both a molecular transporter and as an anchor in vivo. A portion of the molecule C-terminal to the canonical lever arm, termed the medial tail (MT), has been proposed to act as either a lever arm extension or as a dimerization motif. We describe constructs in which the MT is interrupted by a glycine-rich molecular swivel. Disruption of the MT results in decreased processive run lengths measured using single-molecule fluorescence microscopy and a decreased step size under applied load as measured in an optical trap. We used single-molecule gold nanoparticle tracking and optical trapping to examine the mechanism of coordination between the heads of dimeric myosin VI. We detect two rate-limiting kinetic processes at low (< 200 micromolar) ATP concentrations. Our data can be explained by a model in which intramolecular tension greatly increases the affinity of the lead head for ADP, likely by slowing ADP release from the lead head. This mechanism likely increases both the motor's processivity and its ability to act as an anchor under physiological conditions.

    View details for DOI 10.1073/pnas.1002430107

    View details for Web of Science ID 000277088700028

    View details for PubMedID 20385849

    View details for PubMedCentralID PMC2867888

  • Engineered Myosin VI Motors Reveal Minimal Structural Determinants of Directionality and Processivity JOURNAL OF MOLECULAR BIOLOGY Liao, J., Elting, M. W., Delp, S. L., Spudich, J. A., Bryant, Z. 2009; 392 (4): 862-867

    Abstract

    Myosins have diverse mechanical properties reflecting a range of cellular roles. A major challenge is to understand the structural basis for generating novel functions from a common motor core. Myosin VI (M6) is specialized for processive motion toward the (-) end of actin filaments. We have used engineered M6 motors to test and refine the "redirected power stroke" model for (-) end directionality and to explore poorly understood structural requirements for processive stepping. Guided by crystal structures and molecular modeling, we fused artificial lever arms to the catalytic head of M6 at several positions, retaining varying amounts of native structure. We found that an 18-residue alpha-helical insert is sufficient to reverse the directionality of the motor, with no requirement for any calmodulin light chains. Further, we observed robust processive stepping of motors with artificial lever arms, demonstrating that processivity can arise without optimizing lever arm composition or mechanics.

    View details for DOI 10.1016/j.jmb.2009.07.046

    View details for Web of Science ID 000270601200002

    View details for PubMedID 19631216

    View details for PubMedCentralID PMC3360974

  • Coarse-Grained Structural Modeling of Molecular Motors Using Multibody Dynamics CELLULAR AND MOLECULAR BIOENGINEERING Parker, D., Bryant, Z., Delp, S. L. 2009; 2 (3): 366-374

    Abstract

    Experimental and computational approaches are needed to uncover the mechanisms by which molecular motors convert chemical energy into mechanical work. In this article, we describe methods and software to generate structurally realistic models of molecular motor conformations compatible with experimental data from different sources. Coarse-grained models of molecular structures are constructed by combining groups of atoms into a system of rigid bodies connected by joints. Contacts between rigid bodies enforce excluded volume constraints, and spring potentials model system elasticity. This simplified representation allows the conformations of complex molecular motors to be simulated interactively, providing a tool for hypothesis building and quantitative comparisons between models and experiments. In an example calculation, we have used the software to construct atomically detailed models of the myosin V molecular motor bound to its actin track. The software is available at www.simtk.org.

    View details for DOI 10.1007/s12195-009-0084-4

    View details for Web of Science ID 000270168900010

    View details for PubMedCentralID PMC2860290

  • Coarse-Grained Structural Modeling of Molecular Motors Using Multibody Dynamics. Cellular and molecular bioengineering Parker, D., Bryant, Z., Delp, S. L. 2009; 2 (3): 366?74

    Abstract

    Experimental and computational approaches are needed to uncover the mechanisms by which molecular motors convert chemical energy into mechanical work. In this article, we describe methods and software to generate structurally realistic models of molecular motor conformations compatible with experimental data from different sources. Coarse-grained models of molecular structures are constructed by combining groups of atoms into a system of rigid bodies connected by joints. Contacts between rigid bodies enforce excluded volume constraints, and spring potentials model system elasticity. This simplified representation allows the conformations of complex molecular motors to be simulated interactively, providing a tool for hypothesis building and quantitative comparisons between models and experiments. In an example calculation, we have used the software to construct atomically detailed models of the myosin V molecular motor bound to its actin track. The software is available at www.simtk.org.

    View details for PubMedID 20428469

  • Multiple modes of Escherichia coli DNA gyrase activity revealed by force and torque NATURE STRUCTURAL & MOLECULAR BIOLOGY Nollmann, M., Stone, M. D., Bryant, Z., Gore, J., Crisona, N. J., Hong, S., Mitelheiser, S., Maxwell, A., Bustamante, C., Cozzarelli, N. R. 2007; 14 (4): 264-271

    Abstract

    E. coli DNA gyrase uses the energy of ATP hydrolysis to introduce essential negative supercoils into the genome, thereby working against the mechanical stresses that accumulate in supercoiled DNA. Using a magnetic-tweezers assay, we demonstrate that small changes in force and torque can switch gyrase among three distinct modes of activity. Under low mechanical stress, gyrase introduces negative supercoils by a mechanism that depends on DNA wrapping. Elevated tension or positive torque suppresses DNA wrapping, revealing a second mode of activity that resembles the activity of topoisomerase IV. This 'distal T-segment capture' mode results in active relaxation of left-handed braids and positive supercoils. A third mode is responsible for the ATP-independent relaxation of negative supercoils. We present a branched kinetic model that quantitatively accounts for all of our single-molecule results and agrees with existing biochemical data.

    View details for DOI 10.1038/nsmb1213

    View details for Web of Science ID 000245469600011

    View details for PubMedID 17334374

  • The power stroke of myosin VI and the basis of reverse directionality PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bryant, Z., Altman, D., Spudich, J. A. 2007; 104 (3): 772-777

    Abstract

    Myosin VI supports movement toward the (-) end of actin filaments, despite sharing extensive sequence and structural homology with (+)-end-directed myosins. A class-specific stretch of amino acids inserted between the converter domain and the lever arm was proposed to provide the structural basis of directionality reversal. Indeed, the unique insert mediates a 120 degrees redirection of the lever arm in a crystal structure of the presumed poststroke conformation of myosin VI [Ménétrey J, Bahloul A, Wells AL, Yengo CM, Morris CA, Sweeney HL, Houdusse A (2005) Nature 435:779-785]. However, this redirection alone is insufficient to account for the large (-)-end-directed stroke of a monomeric myosin VI construct. The underlying motion of the myosin VI converter domain must therefore differ substantially from the power stroke of (+)-end-directed myosins. To experimentally map out the motion of the converter domain and lever arm, we have generated a series of truncated myosin VI constructs and characterized the size and direction of the power stroke for each construct using dual-labeled gliding filament assays and optical trapping. Motors truncated near the end of the converter domain generate (+)-end-directed motion, whereas longer constructs move toward the (-) end. Our results directly demonstrate that the unique insert is required for directionality reversal, ruling out a large class of models in which the converter domain moves toward the (-) end. We suggest that the lever arm rotates approximately 180 degrees between pre- and poststroke conformations.

    View details for DOI 10.1073/pnas.0610144104

    View details for Web of Science ID 000243761100018

    View details for PubMedID 17182734

    View details for PubMedCentralID PMC1713167

  • DNA overwinds when stretched NATURE Gore, J., Bryant, Z., Noellmann, M., Le, M. U., Cozzarelli, N. R., Bustamante, C. 2006; 442 (7104): 836-839

    Abstract

    DNA is often modelled as an isotropic rod, but its chiral structure suggests the possible importance of anisotropic mechanical properties, including coupling between twisting and stretching degrees of freedom. Simple physical intuition predicts that DNA should unwind under tension, as it is pulled towards a denatured structure. We used rotor bead tracking to directly measure twist-stretch coupling in single DNA molecules. Here we show that for small distortions, contrary to intuition, DNA overwinds under tension, reaching a maximum twist at a tension of approximately 30 pN. As tension is increased above this critical value, the DNA begins to unwind. The observed twist-stretch coupling predicts that DNA should also lengthen when overwound under constant tension, an effect that we quantitatively confirm. We present a simple model that explains these unusual mechanical properties, and also suggests a possible origin for the anomalously large torsional rigidity of DNA. Our results have implications for the action of DNA-binding proteins that must stretch and twist DNA to compensate for variability in the lengths of their binding sites. The requisite coupled DNA distortions are favoured by the intrinsic mechanical properties of the double helix reported here.

    View details for DOI 10.1038/nature04974

    View details for Web of Science ID 000239792700051

    View details for PubMedID 16862122

  • Mechanochemical analysis of DNA gyrase using rotor bead tracking NATURE Gore, J., Bryant, Z., Stone, M. D., Nollmann, M. N., Cozzarelli, N. R., Bustamante, C. 2006; 439 (7072): 100-104

    Abstract

    DNA gyrase is a molecular machine that uses the energy of ATP hydrolysis to introduce essential negative supercoils into DNA. The directionality of supercoiling is ensured by chiral wrapping of the DNA around a specialized domain of the enzyme before strand passage. Here we observe the activity of gyrase in real time by tracking the rotation of a submicrometre bead attached to the side of a stretched DNA molecule. In the presence of gyrase and ATP, we observe bursts of rotation corresponding to the processive, stepwise introduction of negative supercoils in strict multiples of two. Changes in DNA tension have no detectable effect on supercoiling velocity, but the enzyme becomes markedly less processive as tension is increased over a range of only a few tenths of piconewtons. This behaviour is quantitatively explained by a simple mechanochemical model in which processivity depends on a kinetic competition between dissociation and rapid, tension-sensitive DNA wrapping. In a high-resolution variant of our assay, we directly detect rotational pauses corresponding to two kinetic substeps: an ATP-independent step at the end of the reaction cycle, and an ATP-binding step in the middle of the cycle, subsequent to DNA wrapping.

    View details for DOI 10.1038/nature04319

    View details for Web of Science ID 000234378700042

    View details for PubMedID 16397501

    View details for PubMedCentralID PMC1440892

  • Chirality sensing by Escherichia coli topoisomerase IV and the mechanism of type II topoisomerases PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Stone, M. D., Bryant, Z., Crisona, N. J., Smith, S. B., Vologodskii, A., Bustamante, C., Cozzarelli, N. R. 2003; 100 (15): 8654-8659

    Abstract

    Escherichia coli topoisomerase (Topo) IV is an essential type II Topo that removes DNA entanglements created during DNA replication. Topo IV relaxes (+) supercoils much faster than (-) supercoils, promoting replication while sparing the essential (-) supercoils. Here, we investigate the mechanism underlying this chiral preference. Using DNA binding assays and a single-molecule DNA braiding system, we show that Topo IV recognizes the chiral crossings imposed by the left-handed superhelix of a (+) supercoiled DNA, rather than global topology, twist deformation, or local writhe. Monte Carlo simulations of braid, supercoil, and catenane configurations demonstrate how a preference for a single-crossing geometry during strand passage can allow Topo IV to perform its physiological functions. Single-enzyme braid relaxation experiments also provide a direct measure of the processivity of the enzyme and offer insight into its mechanochemical cycle.

    View details for DOI 10.1073/pnas.1133178100

    View details for Web of Science ID 000184371000013

    View details for PubMedID 12857958

  • Structural transitions and elasticity from torque measurements on DNA NATURE Bryant, Z., Stone, M. D., Gore, J., Smith, S. B., Cozzarelli, N. R., Bustamante, C. 2003; 424 (6946): 338-341

    Abstract

    Knowledge of the elastic properties of DNA is required to understand the structural dynamics of cellular processes such as replication and transcription. Measurements of force and extension on single molecules of DNA have allowed direct determination of the molecule's mechanical properties, provided rigorous tests of theories of polymer elasticity, revealed unforeseen structural transitions induced by mechanical stresses, and established an experimental and conceptual framework for mechanical assays of enzymes that act on DNA. However, a complete description of DNA mechanics must also consider the effects of torque, a quantity that has hitherto not been directly measured in micromanipulation experiments. We have measured torque as a function of twist for stretched DNA--torsional strain in over- or underwound molecules was used to power the rotation of submicrometre beads serving as calibrated loads. Here we report tests of the linearity of DNA's twist elasticity, direct measurements of the torsional modulus (finding a value approximately 40% higher than generally accepted), characterization of torque-induced structural transitions, and the establishment of a framework for future assays of torque and twist generation by DNA-dependent enzymes. We also show that cooperative structural transitions in DNA can be exploited to construct constant-torque wind-up motors and force-torque converters.

    View details for DOI 10.1038/nature01810

    View details for Web of Science ID 000184183900048

    View details for PubMedID 12867987

  • Ten years of tension: single-molecule DNA mechanics NATURE Bustamante, C., Bryant, Z., Smith, S. B. 2003; 421 (6921): 423-427

    Abstract

    The basic features of DNA were elucidated during the half-century following the discovery of the double helix. But it is only during the past decade that researchers have been able to manipulate single molecules of DNA to make direct measurements of its mechanical properties. These studies have illuminated the nature of interactions between DNA and proteins, the constraints within which the cellular machinery operates, and the forces created by DNA-dependent motors.

    View details for DOI 10.1038/nature01405

    View details for Web of Science ID 000180533000056

    View details for PubMedID 12540915

  • Conjugation of DNA to silanized colloidal semiconductor nanocrystalline quantum dots CHEMISTRY OF MATERIALS Parak, W. J., Gerion, D., Zanchet, D., Woerz, A. S., Pellegrino, T., Micheel, C., Williams, S. C., Seitz, M., Bruehl, R. E., Bryant, Z., Bustamante, C., Bertozzi, C. R., Alivisatos, A. P. 2002; 14 (5): 2113-2119

    View details for DOI 10.1021/cm0107878

    View details for Web of Science ID 000175790100030

  • Mechanical unfolding of a beta-hairpin using molecular dynamics BIOPHYSICAL JOURNAL Bryant, Z., Pande, V. S., Rokhsar, D. S. 2000; 78 (2): 584-589

    Abstract

    Single-molecule mechanical unfolding experiments have the potential to provide insights into the details of protein folding pathways. To investigate the relationship between force-extension unfolding curves and microscopic events, we performed molecular dynamics simulations of the mechanical unfolding of the C-terminal hairpin of protein G. We have studied the dependence of the unfolding pathway on pulling speed, cantilever stiffness, and attachment points. Under conditions that generate low forces, the unfolding trajectory mimics the untethered, thermally accessible pathway previously proposed based on high-temperature studies. In this stepwise pathway, complete breakdown of backbone hydrogen bonds precedes dissociation of the hydrophobic cluster. Under more extreme conditions, the cluster and hydrogen bonds break simultaneously. Transitions between folding intermediates can be identified in our simulations as features of the calculated force-extension curves.

    View details for Web of Science ID 000085249300004

    View details for PubMedID 10653773

  • Characterization of differentially expressed genes in purified Drosophila follicle cells: Toward a general strategy for cell type-specific developmental analysis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bryant, Z., Subrahmanyan, L., Tworoger, M., LaTray, L., Liu, C. R., Li, M. J., van den Engh, G., Ruohola-Baker, H. 1999; 96 (10): 5559-5564

    Abstract

    Axis formation in Drosophila depends on correct patterning of the follicular epithelium and on signaling between the germ line and soma during oogenesis. We describe a method for identifying genes expressed in the follicle cells with potential roles in axis formation. Follicle cells are purified from whole ovaries by enzymatic digestion, filtration, and fluorescence-activated cell sorting (FACS). Two strategies are used to obtain complementary cell groups. In the first strategy, spatially restricted subpopulations are marked for FACS selection using a green fluorescent protein (GFP) reporter. In the second, cells are purified from animals mutant for the epidermal growth factor receptor ligand gurken (grk) and from their wild-type siblings. cDNA from these samples of spatially restricted or genetically mutant follicle cells is used in differential expression screens employing PCR-based differential display or hybridization to a cDNA microarray. Positives are confirmed by in situ hybridization to whole mounts. These methods are found to be capable of identifying both spatially restricted and grk-dependent transcripts. Results from our pilot screens include (i) the identification of a homologue of the immunophilin FKBP-12 with dorsal anterior expression in egg chambers, (ii) the discovery that the ecdysone-inducible nuclear hormone receptor gene E78 is regulated by grk during oogenesis and is required for proper dorsal appendage formation, and (iii) the identification of a Drosophila homologue of the human SET-binding factor gene SBF1 with elevated transcription in grk mutant egg chambers.

    View details for Web of Science ID 000080246500045

    View details for PubMedID 10318923

  • Mosaic analysis in the Drosophila ovary reveals a common hedgehog-inducible precursor stage for stalk and polar cells GENETICS Tworoger, M., Larkin, M. K., Bryant, Z., Ruohola-Baker, H. 1999; 151 (2): 739-748

    Abstract

    The fates of two small subgroups of the ovarian follicle cells appear to be linked: mutations in Notch, Delta, fs(1)Yb, or hedgehog cause simultaneous defects in the specification of stalk cells and polar cells. Both of these subgroups are determined in the germarium, and both cease division early in oogenesis. To test the possibility that these subgroups are related by lineage, we generated dominantly marked mitotic clones in ovaries. Small, restricted clones in stalk cells and polar cells were found adjacent to each other at a frequency much too high to be explained by independent induction. We therefore propose a model in which stalk cells and polar cells are derived from a precursor population that is distinct from the precursors for other follicle cells. We support and extend this model by characterization of mutants that affect stalk and polar cell formation. We find that ectopic expression of Hedgehog can induce both polar and stalk cell fate, presumably by acting on the precursor stage. In contrast, we find that stall affects neither the induction of the precursors nor the decision between the stalk cell and polar cell fate but, rather, some later differentiation step of stalk cells. In addition, we show that ectopic polar and stalk cells disturb the anterior-posterior polarity of the underlying oocyte.

    View details for Web of Science ID 000078462500027

    View details for PubMedID 9927465

  • maelstrom is required for an early step in the establishment of Drosophila oocyte polarity: posterior localization of grk mRNA DEVELOPMENT Clegg, N. J., Frost, D. M., Larkin, M. K., Subrahmanyan, L., Bryant, Z., Ruohola-Baker, H. 1997; 124 (22): 4661-4671

    Abstract

    We describe a mutant, maelstrom, that disrupts a previously unobserved step in mRNA localization within the early oocyte, distinct from nurse-cell-to-oocyte RNA transport. Mutations in maelstrom disturb the localization of mRNAs for Gurken (a ligand for the Drosophila Egf receptor), Oskar and Bicoid at the posterior of the developing (stage 3-6) oocyte. maelstrom mutants display phenotypes detected in gurken loss-of-function mutants: posterior follicle cells with anterior cell fates, bicoid mRNA localization at both poles of the stage 8 oocyte and ventralization of the eggshell. These data are consistent with the suggestion that early posterior localization of gurken mRNA is essential for activation of the Egf receptor pathway in posterior follicle cells. Posterior localization of mRNA in stage 3-6 oocytes could therefore be one of the earliest known steps in the establishment of oocyte polarity. The maelstrom gene encodes a novel protein that has a punctate distribution in the cytoplasm of the nurse cells and the oocyte until the protein disappears in stage 7 of oogenesis.

    View details for Web of Science ID 000071111800023

    View details for PubMedID 9409682

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