Bio

Academic Appointments


Honors & Awards


  • Member, National Academy of Sciences (elected 2007)
  • Member, American Academy of Arts & Sciences (elected 2000)
  • Fellow, American Association for the Advancement of Science (elected 2006)
  • National Lecturer, Biophysical Society (2012)
  • MERIT Award, NIH-NIGMS (2010-2015)
  • Max Delbruck Prize in Biological Physics, American Physical Society (2008)
  • Outstanding Investigator Award in Single Molecule Biophysics, Biophysical Society (2008)
  • Research Excellence Award, U. Penn Nano/Bio Interface Center (2007)
  • S.W. Ascherman Chair of Sciences, Stanford University (2006)
  • President, Biophysical Society (2005-2006)
  • Young Investigator Award, Biophysical Society (1994)

Professional Education


  • PhD, California Inst. of Technology, Biology (1983)
  • MA, Univ. Colorado, Biology (1982)
  • MA, Oxford University, UK, Physics (1978)
  • BA, Oxford University, UK, Physics (1974)

Research & Scholarship

Current Research and Scholarly Interests


Single molecule biophysics using optical trapping and fluorescence

Teaching

2013-14 Courses


Postdoctoral Advisees


Graduate and Fellowship Programs


Publications

Journal Articles


  • Binding and Translocation of Termination Factor Rho Studied at the Single-Molecule Level JOURNAL OF MOLECULAR BIOLOGY Koslover, D. J., Fazal, F. M., Mooney, R. A., Landick, R., Block, S. M. 2012; 423 (5): 664-676

    Abstract

    Rho termination factor is an essential hexameric helicase responsible for terminating 20-50% of all mRNA synthesis in Escherichia coli. We used single-molecule force spectroscopy to investigate Rho-RNA binding interactions at the Rho utilization site of the ?tR1 terminator. Our results are consistent with Rho complexes adopting two states: one that binds 57 ± 2nt of RNA across all six of the Rho primary binding sites, and another that binds 85 ± 2nt at the six primary sites plus a single secondary site situated at the center of the hexamer. The single-molecule data serve to establish that Rho translocates 5'?3' toward RNA polymerase (RNAP) by a tethered-tracking mechanism, looping out the intervening RNA between the Rho utilization site and RNAP. These findings lead to a general model for Rho binding and translocation and establish a novel experimental approach that should facilitate additional single-molecule studies of RNA-binding proteins.

    View details for DOI 10.1016/j.jmb.2012.07.027

    View details for Web of Science ID 000310666400002

    View details for PubMedID 22885804

  • Direct Observation of Cotranscriptional Folding in an Adenine Riboswitch SCIENCE Frieda, K. L., Block, S. M. 2012; 338 (6105): 397-400

    Abstract

    Growing RNA chains fold cotranscriptionally as they are synthesized by RNA polymerase. Riboswitches, which regulate gene expression by adopting alternative RNA folds, are sensitive to cotranscriptional events. We developed an optical-trapping assay to follow the cotranscriptional folding of a nascent RNA and used it to monitor individual transcripts of the pbuE adenine riboswitch, visualizing distinct folding transitions. We report a particular folding signature for the riboswitch aptamer whose presence directs the gene-regulatory transcription outcome, and we measured the termination frequency as a function of adenine level and tension applied to the RNA. Our results demonstrate that the outcome is kinetically controlled. These experiments furnish a means to observe conformational switching in real time and enable the precise mapping of events during cotranscriptional folding.

    View details for DOI 10.1126/science.1225722

    View details for Web of Science ID 000309955800045

    View details for PubMedID 23087247

  • A universal pathway for kinesin stepping NATURE STRUCTURAL & MOLECULAR BIOLOGY Clancy, B. E., Behnke-Parks, W. M., Andreasson, J. O., Rosenfeld, S. S., Block, S. M. 2011; 18 (9): 1020-U79

    Abstract

    Kinesin-1 is an ATP-driven, processive motor that transports cargo along microtubules in a tightly regulated stepping cycle. Efficient gating mechanisms ensure that the sequence of kinetic events proceeds in the proper order, generating a large number of successive reaction cycles. To study gating, we created two mutant constructs with extended neck-linkers and measured their properties using single-molecule optical trapping and ensemble fluorescence techniques. Owing to a reduction in the inter-head tension, the constructs access an otherwise rarely populated conformational state in which both motor heads remain bound to the microtubule. ATP-dependent, processive backstepping and futile hydrolysis were observed under moderate hindering loads. On the basis of measurements, we formulated a comprehensive model for kinesin motion that incorporates reaction pathways for both forward and backward stepping. In addition to inter-head tension, we found that neck-linker orientation is also responsible for ensuring gating in kinesin.

    View details for DOI 10.1038/nsmb.2104

    View details for Web of Science ID 000294551200010

    View details for PubMedID 21841789

  • Applied force reveals mechanistic and energetic details of transcription termination CELL Larson, M. H., Greenleaf, W. J., Landick, R., Block, S. M. 2008; 132 (6): 971-982

    Abstract

    Transcription termination by bacterial RNA polymerase (RNAP) occurs at sequences coding for a GC-rich RNA hairpin followed by a U-rich tract. We used single-molecule techniques to investigate the mechanism by which three representative terminators (his, t500, and tR2) destabilize the elongation complex (EC). For his and tR2 terminators, loads exerted to bias translocation did not affect termination efficiency (TE). However, the force-dependent kinetics of release and the force-dependent TE of a mutant imply a forward translocation mechanism for the t500 terminator. Tension on isolated U-tracts induced transcript release in a manner consistent with RNA:DNA hybrid shearing. We deduce that different mechanisms, involving hypertranslocation or shearing, operate at terminators with different U-tracts. Tension applied to RNA at terminators suggests that closure of the final 2-3 hairpin bases destabilizes the hybrid and that competing RNA structures modulate TE. We propose a quantitative, energetic model that predicts the behavior for these terminators and mutant variants.

    View details for DOI 10.1016/j.cell.2008.01.027

    View details for Web of Science ID 000254273600016

    View details for PubMedID 18358810

  • Direct observation of hierarchical folding in single riboswitch aptamers SCIENCE Greenleaf, W. J., Frieda, K. L., Foster, D. A., Woodside, M. T., Block, S. M. 2008; 319 (5863): 630-633

    Abstract

    Riboswitches regulate genes through structural changes in ligand-binding RNA aptamers. With the use of an optical-trapping assay based on in situ transcription by a molecule of RNA polymerase, single nascent RNAs containing pbuE adenine riboswitch aptamers were unfolded and refolded. Multiple folding states were characterized by means of both force-extension curves and folding trajectories under constant force by measuring the molecular contour length, kinetics, and energetics with and without adenine. Distinct folding steps correlated with the formation of key secondary or tertiary structures and with ligand binding. Adenine-induced stabilization of the weakest helix in the aptamer, the mechanical switch underlying regulatory action, was observed directly. These results provide an integrated view of hierarchical folding in an aptamer, demonstrating how complex folding can be resolved into constituent parts, and supply further insights into tertiary structure formation.

    View details for DOI 10.1126/science.1151298

    View details for Web of Science ID 000252772000044

    View details for PubMedID 18174398

  • Direct measurement of the full, sequence-dependent folding landscape of a nucleic acid SCIENCE Woodside, M. T., Anthony, P. C., Behnke-Parks, W. M., Larizadeh, K., Herschlag, D., Block, S. M. 2006; 314 (5801): 1001-1004

    Abstract

    Nucleic acid hairpins provide a powerful model system for understanding macromolecular folding, with free-energy landscapes that can be readily manipulated by changing the hairpin sequence. The full shapes of energy landscapes for the reversible folding of DNA hairpins under controlled loads exerted by an optical force clamp were obtained by deconvolution from high-resolution, single-molecule trajectories. The locations and heights of the energy barriers for hairpin folding could be tuned by adjusting the number and location of G:C base pairs, and the presence and position of folding intermediates were controlled by introducing single-nucleotide mismatches.

    View details for DOI 10.1126/science.1133601

    View details for Web of Science ID 000241896000056

    View details for PubMedID 17095702

  • Sequence-resolved detecton of pausing by single RNA polymerase molecules CELL Herbert, K. M., La Porta, A., Wong, B. J., Mooney, R. A., Neuman, K. C., Landick, R., Block, S. M. 2006; 125 (6): 1083-1094

    Abstract

    Transcriptional pausing by RNA polymerase (RNAP) plays an important role in the regulation of gene expression. Defined, sequence-specific pause sites have been identified biochemically. Single-molecule studies have also shown that bacterial RNAP pauses frequently during transcriptional elongation, but the relationship of these "ubiquitous" pauses to the underlying DNA sequence has been uncertain. We employed an ultrastable optical-trapping assay to follow the motion of individual molecules of RNAP transcribing templates engineered with repeated sequences carrying imbedded, sequence-specific pause sites of known regulatory function. Both the known and ubiquitous pauses appeared at reproducible locations, identified with base-pair accuracy. Ubiquitous pauses were associated with DNA sequences that show similarities to regulatory pause sequences. Data obtained for the lifetimes and efficiencies of pauses support a model where the transition to pausing branches off of the normal elongation pathway and is mediated by a common elemental state, which corresponds to the ubiquitous pause.

    View details for DOI 10.1016/j.cell.2006.04.032

    View details for Web of Science ID 000238602700014

    View details for PubMedID 16777599

  • Direct observation of base-pair stepping by RNA polymerase NATURE Abbondanzieri, E. A., Greenleaf, W. J., Shaevitz, J. W., Landick, R., Block, S. M. 2005; 438 (7067): 460-465

    Abstract

    During transcription, RNA polymerase (RNAP) moves processively along a DNA template, creating a complementary RNA. Here we present the development of an ultra-stable optical trapping system with ångström-level resolution, which we used to monitor transcriptional elongation by single molecules of Escherichia coli RNAP. Records showed discrete steps averaging 3.7 +/- 0.6 A, a distance equivalent to the mean rise per base found in B-DNA. By combining our results with quantitative gel analysis, we conclude that RNAP advances along DNA by a single base pair per nucleotide addition to the nascent RNA. We also determined the force-velocity relationship for transcription at both saturating and sub-saturating nucleotide concentrations; fits to these data returned a characteristic distance parameter equivalent to one base pair. Global fits were inconsistent with a model for movement incorporating a power stroke tightly coupled to pyrophosphate release, but consistent with a brownian ratchet model incorporating a secondary NTP binding site.

    View details for DOI 10.1038/nature04268

    View details for Web of Science ID 000233458200041

    View details for PubMedID 16284617

  • Single-molecule studies of RNAPII elongation BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS Zhou, J., Schweikhard, V., Block, S. M. 2013; 1829 (1): 29-38

    Abstract

    Elongation, the transcriptional phase in which RNA polymerase (RNAP) moves processively along a DNA template, occurs via a fundamental enzymatic mechanism that is thought to be universally conserved among multi-subunit polymerases in all kingdoms of life. Beyond this basic mechanism, a multitude of processes are integrated into transcript elongation, among them fidelity control, gene regulatory interactions involving elongation factors, RNA splicing or processing factors, and regulatory mechanisms associated with chromatin structure. Many kinetic and molecular details of the mechanism of the nucleotide addition cycle and its regulation, however, remain elusive and generate continued interest and even controversy. Recently, single-molecule approaches have emerged as powerful tools for the study of transcription in eukaryotic organisms. Here, we review recent progress and discuss some of the unresolved questions and ongoing debates, while anticipating future developments in the field. This article is part of a Special Issue entitled: RNA Polymerase II Transcript Elongation.

    View details for DOI 10.1016/j.bbagrm.2012.08.006

    View details for Web of Science ID 000314616600005

    View details for PubMedID 22982192

  • Trigger loop dynamics mediate the balance between the transcriptional fidelity and speed of RNA polymerase II PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Larson, M. H., Zhou, J., Kaplan, C. D., Palangat, M., Kornberg, R. D., Landick, R., Block, S. M. 2012; 109 (17): 6555-6560

    Abstract

    During transcription, RNA polymerase II (RNAPII) must select the correct nucleotide, catalyze its addition to the growing RNA transcript, and move stepwise along the DNA until a gene is fully transcribed. In all kingdoms of life, transcription must be finely tuned to ensure an appropriate balance between fidelity and speed. Here, we used an optical-trapping assay with high spatiotemporal resolution to probe directly the motion of individual RNAPII molecules as they pass through each of the enzymatic steps of transcript elongation. We report direct evidence that the RNAPII trigger loop, an evolutionarily conserved protein subdomain, serves as a master regulator of transcription, affecting each of the three main phases of elongation, namely: substrate selection, translocation, and catalysis. Global fits to the force-velocity relationships of RNAPII and its trigger loop mutants support a Brownian ratchet model for elongation, where the incoming NTP is able to bind in either the pre- or posttranslocated state, and movement between these two states is governed by the trigger loop. Comparison of the kinetics of pausing by WT and mutant RNAPII under conditions that promote base misincorporation indicate that the trigger loop governs fidelity in substrate selection and mismatch recognition, and thereby controls aspects of both transcriptional accuracy and rate.

    View details for DOI 10.1073/pnas.1200939109

    View details for Web of Science ID 000303249100047

    View details for PubMedID 22493230

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

    Abstract

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

    View details for DOI 10.1021/ja208466h

    View details for Web of Science ID 000301990600036

    View details for PubMedID 22369617

  • Folding energy landscape of the thiamine pyrophosphate riboswitch aptamer PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Anthony, P. C., Perez, C. F., Garcia-Garcia, C., Block, S. M. 2012; 109 (5): 1485-1489

    Abstract

    Riboswitches are motifs in the untranslated regions (UTRs) of RNA transcripts that sense metabolite levels and modulate the expression of the corresponding genes for metabolite import, export, synthesis, or degradation. All riboswitches contain an aptamer: an RNA structure that, upon binding ligand, folds to expose or sequester regulatory elements in the adjacent sequence through alternative nucleotide pairing. The coupling between ligand binding and aptamer folding is central to the regulatory mechanisms of thiamine pyrophosphate (TPP) riboswitches and has not been fully characterized. Here, we show that TPP aptamer folding can be decomposed into ligand-independent and -dependent steps that correspond to the formation of secondary and tertiary structures, respectively. We reconstructed the full energy landscape for folding of the wild-type (WT) aptamer and measured perturbations of this landscape arising from mutations or ligand binding. We show that TPP binding proceeds in two steps, from a weakly to a strongly bound state. Our data imply a hierarchical folding sequence, and provide a framework for understanding molecular mechanism throughout the TPP riboswitch family.

    View details for DOI 10.1073/pnas.1115045109

    View details for Web of Science ID 000299731400035

    View details for PubMedID 22219369

  • Applied Force Provides Insight into Transcriptional Pausing and Its Modulation by Transcription Factor NusA MOLECULAR CELL Zhou, J., Ha, K. S., La Porta, A., Landick, R., Block, S. M. 2011; 44 (4): 635-646

    Abstract

    Transcriptional pausing by RNA polymerase (RNAP) plays an essential role in gene regulation. Pausing is modified by various elongation factors, including prokaryotic NusA, but the mechanisms underlying pausing and NusA function remain unclear. Alternative models for pausing invoke blockade events that precede translocation (on-pathway), enzyme backtracking (off-pathway), or isomerization to a nonbacktracked, elemental pause state (off-pathway). We employed an optical trapping assay to probe the motions of individual RNAP molecules transcribing a DNA template carrying tandem repeats encoding the his pause, subjecting these enzymes to controlled forces. NusA significantly decreased the pause-free elongation rate of RNAP while increasing the probability of entry into short- and long-lifetime pauses, in a manner equivalent to exerting a ~19 pN force opposing transcription. The effects of force and NusA on pause probabilities and lifetimes support a reaction scheme where nonbacktracked, elemental pauses branch off the elongation pathway from the pretranslocated state of RNAP.

    View details for DOI 10.1016/j.molcel.2011.09.018

    View details for Web of Science ID 000297387800014

    View details for PubMedID 22099310

  • Optical tweezers study life under tension NATURE PHOTONICS Fazal, F. M., Block, S. M. 2011; 5 (6): 318-321

    Abstract

    Optical tweezers have become one of the primary weapons in the arsenal of biophysicists, and have revolutionized the new field of single-molecule biophysics. Today's techniques allow high-resolution experiments on biological macromolecules that were mere pipe dreams only a decade ago.

    View details for Web of Science ID 000291089000003

    View details for PubMedID 22145010

  • Single-Molecule Studies of RNA Polymerase: One Singular Sensation, Every Little Step It Takes MOLECULAR CELL Larson, M. H., Landick, R., Block, S. M. 2011; 41 (3): 249-262

    Abstract

    Transcription is the first of many biochemical steps that turn the genetic information found in DNA into the proteins responsible for driving cellular processes. In this review, we highlight certain advantages of single-molecule techniques in the study of prokaryotic and eukaryotic transcription, and the specific ways in which these techniques complement conventional, ensemble-based biochemistry. We focus on recent literature, highlighting examples where single-molecule methods have provided fresh insights into mechanism. We also present recent technological advances and outline future directions in the field.

    View details for DOI 10.1016/j.molcel.2011.01.008

    View details for Web of Science ID 000287460500004

    View details for PubMedID 21292158

  • E. coli NusG Inhibits Backtracking and Accelerates Pause-Free Transcription by Promoting Forward Trans location of RNA Polymerase JOURNAL OF MOLECULAR BIOLOGY Herbert, K. M., Zhou, J., Mooney, R. A., La Porta, A., Landick, R., Block, S. M. 2010; 399 (1): 17-30

    Abstract

    NusG is an essential transcription factor in Escherichia coli that is capable of increasing the overall rate of transcription. Transcript elongation by RNA polymerase (RNAP) is frequently interrupted by pauses of varying durations, and NusG is known to decrease the occupancy of at least some paused states. However, it has not been established whether NusG enhances transcription chiefly by (1) increasing the rate of elongation between pauses, (2) reducing the lifetimes of pauses, or (3) reducing the rate of entry into paused states. Here, we studied transcription by single molecules of RNAP under various conditions of ribonucleoside triphosphate concentration, applied load, and temperature, using an optical trapping assay capable of distinguishing pauses as brief as 1 s. We found that NusG increases the rate of elongation, that is, the pause-free velocity along the template. Because pauses are off-pathway states that compete with elongation, we observed a concomitant decrease in the rate of entry into short-lifetime, paused states. The effects on short pauses and elongation were comparatively modest, however. More dramatic was the effect of NusG on suppressing entry into long-lifetime ("stabilized") pauses. Because a significant fraction of the time required for the transcription of a typical gene may be occupied by long pauses, NusG is capable of exerting a significant modulatory effect on the rates of RNA synthesis. The observed properties of NusG were consistent with a unified model where the function of this accessory factor is to promote transcriptionally downstream motion of the enzyme along the DNA template, which has the effect of forward-biasing RNAP from the pre-translocated state toward the post-translocated state.

    View details for DOI 10.1016/j.jmb.2010.03.051

    View details for Web of Science ID 000278779900003

    View details for PubMedID 20381500

  • AN OPTICAL APPARATUS FOR ROTATION AND TRAPPING METHODS IN ENZYMOLOGY, VOL 475: SINGLE MOLECULE TOOLS, PT B Gutierrez-Medina, B., Andreasson, J. O., Greenleaf, W. J., Laporta, A., Block, S. M. 2010; 475: 377-404

    Abstract

    We present details of the design, construction, and testing of a single-beam optical tweezers apparatus capable of measuring and exerting torque, as well as force, on microfabricated, optically anisotropic particles (an "optical torque wrench"). The control of angular orientation is achieved by rotating the linear polarization of a trapping laser with an electro-optic modulator (EOM), which affords improved performance over previous designs. The torque imparted to the trapped particle is assessed by measuring the difference between left- and right-circular components of the transmitted light, and constant torque is maintained by feeding this difference signal back into a custom-designed electronic servo loop. The limited angular range of the EOM (+/-180 degrees ) is extended by rapidly reversing the polarization once a threshold angle is reached, enabling the torque clamp to function over unlimited, continuous rotations at high bandwidth. In addition, we developed particles suitable for rotation in this apparatus using microfabrication techniques. Altogether, the system allows for the simultaneous application of forces (approximately 0.1-100 pN) and torques (approximately 1-10,000 pN nm) in the study of biomolecules. As a proof of principle, we demonstrate how our instrument can be used to study the supercoiling of single DNA molecules.

    View details for DOI 10.1016/S0076-6879(10)75015-1

    View details for Web of Science ID 000280733800015

    View details for PubMedID 20627165

  • UK defence group's structure could limit its usefulness NATURE Block, S. M. 2009; 462 (7275): 847-847

    View details for DOI 10.1038/462847c

    View details for Web of Science ID 000272795400019

    View details for PubMedID 20016574

  • Direct measurements of kinesin torsional properties reveal flexible domains and occasional stalk reversals during stepping PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Gutierrez-Medina, B., Fehr, A. N., Block, S. M. 2009; 106 (40): 17007-17012

    Abstract

    Kinesin is a homodimeric motor with two catalytic heads joined to a stalk via short neck linkers (NLs). We measured the torsional properties of single recombinant molecules by tracking the thermal angular motions of fluorescently labeled beads bound to the C terminus of the stalk. When kinesin heads were immobilized on microtubules (MTs) under varied nucleotide conditions, we observed bounded or unbounded angular diffusion, depending on whether one or both heads were attached to the MT. Free rotation implies that NLs act as swivels. From data on constrained diffusion, we conclude that the coiled-coil stalk domains are approximately 30-fold stiffer than its flexible "hinge" regions. Surprisingly, while tracking processive kinesin motion at low ATP concentrations, we observed occasional abrupt reversals in the directional orientations of the stalk. Our results impose constraints on kinesin walking models and suggest a role for rotational freedom in cargo transport.

    View details for DOI 10.1073/pnas.0907133106

    View details for Web of Science ID 000270537500028

    View details for PubMedID 19805111

  • On the Origin of Kinesin Limping BIOPHYSICAL JOURNAL Fehr, A. N., Gutierrez-Medina, B., Asbury, C. L., Block, S. M. 2009; 97 (6): 1663-1670

    Abstract

    Kinesin is a dimeric motor with twin catalytic heads joined to a common stalk. Kinesin molecules move processively along microtubules in a hand-over-hand walk, with the two heads advancing alternately. Recombinant kinesin constructs with short stalks have been found to "limp", i.e., exhibit alternation in the dwell times of successive steps. Limping behavior implies that the molecular rearrangements underlying even- and odd-numbered steps must differ, but the mechanism by which such rearrangements lead to limping remains unsolved. Here, we used an optical force clamp to measure individual, recombinant dimers and test candidate explanations for limping. Introducing a covalent cross-link into the stalk region near the heads had no effect on limping, ruling out possible stalk misregistration during coiled-coil formation as a cause. Limping was equally unaffected by mutations that produced 50-fold changes in stalk stiffness, ruling out models where limping arises from an asymmetry in torsional strain. However, limping was enhanced by perturbations that increased the vertical component of load on the motor, including increases in bead size or net load, and decreases in the stalk length. These results suggest that kinesin heads take different vertical trajectories during alternate steps, and that the rates for these motions are differentially sensitive to load.

    View details for DOI 10.1016/j.bpj.2009.07.004

    View details for Web of Science ID 000270380800017

    View details for PubMedID 19751671

  • Direct observation of the binding state of the kinesin head to the microtubule NATURE Guydosh, N. R., Block, S. M. 2009; 461 (7260): 125-U137

    Abstract

    The dimeric motor protein kinesin-1 converts chemical energy from ATP hydrolysis into mechanical work used to transport cargo along microtubules. Cargo attached to the kinesin stalk moves processively in 8-nm increments as its twin motor domains (heads) carry out an asymmetric, 'hand-over-hand' walk. The extent of individual head interactions with the microtubule during stepping, however, remains controversial. A major experimental limitation has been the lack of a means to monitor the attachment of an individual head to the microtubule during movement, necessitating indirect approaches. Here we report the development of a single-molecule assay that can directly report head binding in a walking kinesin molecule, and show that only a single head is bound to the microtubule between steps at low ATP concentrations. A bead was linked to one of the two kinesin heads by means of a short DNA tether and used to apply rapidly alternating hindering and assisting loads with an optical trap. The time-dependent difference between forwards and backwards displacements of the bead alternated between two discrete values during stepping, corresponding to those intervals when the linked head adopted a bound or an unbound state. The linked head could only rebind the microtubule once ATP had become bound to its partner head.

    View details for DOI 10.1038/nature08259

    View details for Web of Science ID 000269478800043

    View details for PubMedID 19693012

  • Precision steering of an optical trap by electro-optic deflection OPTICS LETTERS Valentine, M. T., Guydosh, N. R., Gutierrez-Medina, B., Fehr, A. N., Andreasson, J. O., Block, S. M. 2008; 33 (6): 599-601

    Abstract

    We designed, constructed, and tested a single-beam optical trapping instrument employing twin electro-optic deflectors (EODs) to steer the trap in the specimen plane. Compared with traditional instruments based on acousto-optic deflectors (AODs), EOD-based traps offer a significant improvement in light throughput and a reduction in deflection-angle (pointing) errors. These attributes impart improved force and position resolution, making EOD-based traps a promising alternative for high-precision nanomechanical measurements of biomaterials.

    View details for Web of Science ID 000254907500023

    View details for PubMedID 18347722

  • Kinesin steps do not alternate in size BIOPHYSICAL JOURNAL Fehr, A. N., Asbury, C. L., Block, S. M. 2008; 94 (3): L20-L22

    Abstract

    Kinesin is a two-headed motor protein that transports cargo inside cells by moving stepwise on microtubules. Its exact trajectory along the microtubule is unknown: alternative pathway models predict either uniform 8-nm steps or alternating 7- and 9-nm steps. By analyzing single-molecule stepping traces from "limping" kinesin molecules, we were able to distinguish alternate fast- and slow-phase steps and thereby to calculate the step sizes associated with the motions of each of the two heads. We also compiled step distances from nonlimping kinesin molecules and compared these distributions against models predicting uniform or alternating step sizes. In both cases, we find that kinesin takes uniform 8-nm steps, a result that strongly constrains the allowed models.

    View details for DOI 10.1529/biophysj.107.126839

    View details for Web of Science ID 000252243200002

    View details for PubMedID 18083906

  • Single-molecule studies of RNA polymerase: Motoring along ANNUAL REVIEW OF BIOCHEMISTRY Herbert, K. M., Greenleaf, W. J., Block, S. M. 2008; 77: 149-176

    Abstract

    Single-molecule techniques have advanced our understanding of transcription by RNA polymerase (RNAP). A new arsenal of approaches, including single-molecule fluorescence, atomic-force microscopy, magnetic tweezers, and optical traps (OTs) have been employed to probe the many facets of the transcription cycle. These approaches supply fresh insights into the means by which RNAP identifies a promoter, initiates transcription, translocates and pauses along the DNA template, proofreads errors, and ultimately terminates transcription. Results from single-molecule experiments complement the knowledge gained from biochemical and genetic assays by facilitating the observation of states that are otherwise obscured by ensemble averaging, such as those resulting from heterogeneity in molecular structure, elongation rate, or pause propensity. Most studies to date have been performed with bacterial RNAP, but work is also being carried out with eukaryotic polymerase (Pol II) and single-subunit polymerases from bacteriophages. We discuss recent progress achieved by single-molecule studies, highlighting some of the unresolved questions and ongoing debates.

    View details for DOI 10.1146/annurev.biochem.77.073106.100741

    View details for Web of Science ID 000257596800008

    View details for PubMedID 18410247

  • Not so lame after all: Kinesin still walks with a hobbled head JOURNAL OF GENERAL PHYSIOLOGY Guydosh, N. R., Block, S. M. 2007; 130 (5): 441-444

    View details for DOI 10.1085/jgp.200709902

    View details for Web of Science ID 000250772100001

    View details for PubMedID 17968023

  • Kinesin motor mechanics: Binding, stepping, tracking, gating, and limping BIOPHYSICAL JOURNAL Block, S. M. 2007; 92 (9): 2986-2995

    View details for DOI 10.1529/biophysj.106.100677

    View details for Web of Science ID 000245544100008

    View details for PubMedID 17325011

  • High-resolution, single-molecule measurements of biomolecular motion ANNUAL REVIEW OF BIOPHYSICS AND BIOMOLECULAR STRUCTURE Greenleaf, W. J., Woodside, M. T., Block, S. M. 2007; 36: 171-190

    Abstract

    Many biologically important macromolecules undergo motions that are essential to their function. Biophysical techniques can now resolve the motions of single molecules down to the nanometer scale or even below, providing new insights into the mechanisms that drive molecular movements. This review outlines the principal approaches that have been used for high-resolution measurements of single-molecule motion, including centroid tracking, fluorescence resonance energy transfer, magnetic tweezers, atomic force microscopy, and optical traps. For each technique, the principles of operation are outlined, the capabilities and typical applications are examined, and various practical issues for implementation are considered. Extensions to these methods are also discussed, with an eye toward future application to outstanding biological problems.

    View details for DOI 10.1146/annurev.biophys.36.101106.101451

    View details for Web of Science ID 000247773000009

    View details for PubMedID 17328679

  • Single-molecule, motion-based DNA sequencing using RNA polymerase SCIENCE Greenleaf, W. J., Block, S. M. 2006; 313 (5788): 801-801

    Abstract

    We present a method for sequencing DNA that relies on the motion of single RNA polymerase molecules. When a given nucleotide species limits the rate of transcription, polymerase molecules pause at positions corresponding to the rare base. An ultrastable optical trapping apparatus capable of base pair resolution was used to monitor transcription under limiting amounts of each of the four nucleotide species. From the aligned patterns of pauses recorded from as few as four molecules, we determined the DNA sequence. This proof of principle demonstrates that the motion of a processive nucleic acid enzyme may be used to extract sequence information directly from DNA.

    View details for DOI 10.1126/science.1130105

    View details for Web of Science ID 000239671300049

    View details for PubMedID 16902131

  • Pulling on the nascent RNA during transcription does not alter kinetics of elongation or ubiquitous pausing MOLECULAR CELL Dalal, R. V., Larson, M. H., Neuman, K. C., Gelles, J., Landick, R., Block, S. M. 2006; 23 (2): 231-239

    Abstract

    Transcriptional elongation and termination by RNA polymerase (RNAP) are controlled by interactions among the nascent RNA, DNA, and RNAP that comprise the ternary transcription elongation complex (TEC). To probe the effects of cotranscriptionally folded RNA hairpins on elongation as well as the stability of the TEC, we developed a single-molecule assay to monitor RNA elongation by Escherichia coli RNAP molecules while applying controlled loads to the nascent RNA that favor forward translocation. Remarkably, forces up to 30 pN, twice those required to disrupt RNA secondary structure, did not significantly affect enzyme processivity, transcription elongation rates, pause frequencies, or pause lifetimes. These results indicate that ubiquitous transcriptional pausing is not a consequence of the formation of hairpins in the nascent RNA. The ability of the TEC to sustain large loads on the transcript reflects a tight binding of RNA within the TEC and has important implications for models of transcriptional termination.

    View details for DOI 10.1016/j.molcel.2006.06.023

    View details for Web of Science ID 000239647700011

    View details for PubMedID 16857589

  • Backsteps induced by nucleotide analogs suggest the front head of kinesin is gated by strain PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Guydosh, N. R., Block, S. M. 2006; 103 (21): 8054-8059

    Abstract

    The two-headed kinesin motor harnesses the energy of ATP hydrolysis to take 8-nm steps, walking processively along a microtubule, alternately stepping with each of its catalytic heads in a hand-over-hand fashion. Two persistent challenges for models of kinesin motility are to explain how the two heads are coordinated ("gated") and when the translocation step occurs relative to other events in the mechanochemical reaction cycle. To investigate these questions, we used a precision optical trap to measure the single-molecule kinetics of kinesin in the presence of substrate analogs beryllium fluoride or adenylyl-imidodiphosphate. We found that normal stepping patterns were interspersed with long pauses induced by analog binding, and that these pauses were interrupted by short-lived backsteps. After a pause, processive stepping could only resume once the kinesin molecule took an obligatory, terminal backstep, exchanging the positions of its front and rear heads, presumably to allow release of the bound analog from the new front head. Preferential release from the front head implies that the kinetics of the two heads are differentially affected when both are bound to the microtubule, presumably by internal strain that is responsible for the gating. Furthermore, we found that ATP binding was required to reinitiate processive stepping after the terminal backstep. Together, our results support stepping models in which ATP binding triggers the mechanical step and the front head is gated by strain.

    View details for DOI 10.1073/pnas.0600931103

    View details for Web of Science ID 000237853900023

    View details for PubMedID 16698928

  • Individual dimers of the mitotic kinesin motor Eg5 step processively and support substantial loads in vitro NATURE CELL BIOLOGY Valentine, M. T., Fordyce, P. M., Krzysiak, T. C., Gilbert, S. P., Block, S. M. 2006; 8 (5): 470-U89

    Abstract

    Eg5, a member of the kinesin superfamily of microtubule-based motors, is essential for bipolar spindle assembly and maintenance during mitosis, yet little is known about the mechanisms by which it accomplishes these tasks. Here, we used an automated optical trapping apparatus in conjunction with a novel motility assay that employed chemically modified surfaces to probe the mechanochemistry of Eg5. Individual dimers, formed by a recombinant human construct Eg5-513-5His, stepped processively along microtubules in 8-nm increments, with short run lengths averaging approximately eight steps. By varying the applied load (with a force clamp) and the ATP concentration, we found that the velocity of Eg5 was slower and less sensitive to external load than that of conventional kinesin, possibly reflecting the distinct demands of spindle assembly as compared with vesicle transport. The Eg5-513-5His velocity data were described by a minimal, three-state model where a force-dependent transition follows nucleotide binding.

    View details for DOI 10.1038/ncb1394

    View details for Web of Science ID 000237299400010

    View details for PubMedID 16604065

  • Nanomechanical measurements of the sequence-dependent folding landscapes of single nucleic acid hairpins PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Woodside, M. T., Behnke-Parks, W. M., Larizadeh, K., Travers, K., Herschlag, D., Block, S. M. 2006; 103 (16): 6190-6195

    Abstract

    Nucleic acid hairpins provide a powerful model system for probing the formation of secondary structure. We report a systematic study of the kinetics and thermodynamics of the folding transition for individual DNA hairpins of varying stem length, loop length, and stem GC content. Folding was induced mechanically in a high-resolution optical trap using a unique force clamp arrangement with fast response times. We measured 20 different hairpin sequences with quasi-random stem sequences that were 6-30 bp long, polythymidine loops that were 3-30 nt long, and stem GC content that ranged from 0% to 100%. For all hairpins studied, folding and unfolding were characterized by a single transition. From the force dependence of these rates, we determined the position and height of the energy barrier, finding that the transition state for duplex formation involves the formation of 1-2 bp next to the loop. By measuring unfolding energies spanning one order of magnitude, transition rates covering six orders of magnitude, and hairpin opening distances with subnanometer precision, our results define the essential features of the energy landscape for folding. We find quantitative agreement over the entire range of measurements with a hybrid landscape model that combines thermodynamic nearest-neighbor free energies and nanomechanical DNA stretching energies.

    View details for DOI 10.1073/pnas.0511048103

    View details for Web of Science ID 000236999000025

    View details for PubMedID 16606839

  • Eg5 steps it up! CELL DIVISION Valentine, M. T., Fordyce, P. M., Block, S. M. 2006; 1

    Abstract

    Understanding how molecular motors generate force and move microtubules in mitosis is essential to understanding the physical mechanism of cell division. Recent measurements have shown that one mitotic kinesin superfamily member, Eg5, is mechanically processive and capable of crosslinking and sliding microtubules in vitro. In this review, we highlight recent work that explores how Eg5 functions under load, with an emphasis on the nanomechanical properties of single enzymes.

    View details for DOI 10.1186/1747-1028-1-31

    View details for Web of Science ID 000207723600031

    View details for PubMedID 17173688

  • Picocalorimetry of transcription by RNA polymerase BIOPHYSICAL JOURNAL Abbondanzieri, E. A., Shaevitz, J. W., Block, S. M. 2005; 89 (6): L61-L63

    Abstract

    Thermal variations can exert dramatic effects on the rates of enzymes. The influence of temperature on RNA polymerase is of particular interest because its transcriptional activity governs general levels of gene expression, and may therefore exhibit pleiotropic effects in cells. Using a custom-modified optical trapping apparatus, we used a tightly focused infrared laser to heat single molecules of Escherichia coli RNA polymerase while monitoring transcriptional activity. We found a significant change in rates of transcript elongation with temperature, consistent with a large enthalpic barrier to the condensation reaction associated with RNA polymerization (approximately 13 kcal/mol). In contrast, we found little change in either the frequency or the lifetime of off-pathway, paused states, indicating that the energetic barrier to transcriptional pausing is predominantly entropic.

    View details for DOI 10.1529/biophysj.105.074195

    View details for Web of Science ID 000233590800005

    View details for PubMedID 16239336

  • Passive all-optical force clamp for high-resolution laser trapping PHYSICAL REVIEW LETTERS Greenleaf, W. J., Woodside, M. T., Abbondanzieri, E. A., Block, S. M. 2005; 95 (20)

    Abstract

    Optical traps are useful for studying the effects of forces on single molecules. Feedback-based force clamps are often used to maintain a constant load, but the response time of the feedback limits bandwidth and can introduce instability. We developed a novel force clamp that operates without feedback, taking advantage of the anharmonic region of the trapping potential where the differential stiffness vanishes. We demonstrate the utility of such a force clamp by measuring the unfolding of DNA hairpins and the effect of trap stiffness on opening distance and transition rates.

    View details for DOI 10.1103/PhysRevLett.95.208102

    View details for Web of Science ID 000233243500069

    View details for PubMedID 16384102

  • Statistical kinetics of macromolecular dynamics BIOPHYSICAL JOURNAL Shaevitz, J. W., Block, S. M., Schnitzer, M. J. 2005; 89 (4): 2277-2285

    Abstract

    Fluctuations in biochemical processes can provide insights into the underlying kinetics beyond what can be gleaned from studies of average rates alone. Historically, analysis of fluctuating transmembrane currents supplied information about ion channel conductance states and lifetimes before single-channel recording techniques emerged. More recently, fluctuation analysis has helped to define mechanochemical pathways and coupling ratios for the motor protein kinesin as well as to probe the contributions of static and dynamic disorder to the kinetics of single enzymes. As growing numbers of assays are developed for enzymatic or folding behaviors of single macromolecules, the range of applications for fluctuation analysis increases. To evaluate specific biochemical models against experimental data, one needs to predict analytically the distribution of times required for completion of each reaction pathway. Unfortunately, using traditional methods, such calculations can be challenging for pathways of even modest complexity. Here, we derive an exact expression for the distribution of completion times for an arbitrary pathway with a finite number of states, using a recursive method to solve algebraically for the appropriate moment-generating function. To facilitate comparisons with experiments on processive motor proteins, we develop a theoretical formalism for the randomness parameter, a dimensionless measure of the variance in motor output. We derive the randomness for motors that take steps of variable sizes or that move on heterogeneous substrates, and then discuss possible applications to enzymes such as RNA polymerase, which transcribes varying DNA sequences, and to myosin V and cytoplasmic dynein, which may advance by variable increments.

    View details for DOI 10.1529/biophysj.105.064295

    View details for Web of Science ID 000232147600011

    View details for PubMedID 16040752

  • Measurement of the effective focal shift in an optical trap OPTICS LETTERS Neuman, K. C., Abbondanzieri, E. A., Block, S. M. 2005; 30 (11): 1318-1320

    Abstract

    The focus of an oil-immersion microscope objective is shifted because of the refractive-index mismatch between the cover glass and the aqueous sample. We present a procedure with which to determine the focal shift by use of an inverted microscope equipped with optical tweezers. As the position of the sample chamber is scanned vertically, we measure the axial displacement of an optically trapped bead; the relative motion of the bead with respect to the surface supplies the effective focal shift. Measurements of this quantity deviate from electromagnetic calculations of the focal shift, a discrepancy attributable to the depth-dependent decrease in axial trap stiffness that arises from spherical aberration.

    View details for Web of Science ID 000229259500022

    View details for PubMedID 15981519

  • Simultaneous, coincident optical trapping and single-molecule fluorescence NATURE METHODS Lang, M. J., Fordyce, P. M., Engh, A. M., Neuman, K. C., Block, S. M. 2004; 1 (2): 133-139

    Abstract

    We constructed a microscope-based instrument capable of simultaneous, spatially coincident optical trapping and single-molecule fluorescence. The capabilities of this apparatus were demonstrated by studying the force-induced strand separation of a dye-labeled, 15-base-pair region of double-stranded DNA (dsDNA), with force applied either parallel ('unzipping' mode) or perpendicular ('shearing' mode) to the long axis of the region. Mechanical transitions corresponding to DNA hybrid rupture occurred simultaneously with discontinuous changes in the fluorescence emission. The rupture force was strongly dependent on the direction of applied force, indicating the existence of distinct unbinding pathways for the two force-loading modes. From the rupture force histograms, we determined the distance to the thermodynamic transition state and the thermal off rates in the absence of load for both processes.

    View details for DOI 10.1038/NMETH714

    View details for Web of Science ID 000226753800017

    View details for PubMedID 15782176

  • Optical trapping REVIEW OF SCIENTIFIC INSTRUMENTS Neuman, K. C., Block, S. M. 2004; 75 (9): 2787-2809

    Abstract

    Since their invention just over 20 years ago, optical traps have emerged as a powerful tool with broad-reaching applications in biology and physics. Capabilities have evolved from simple manipulation to the application of calibrated forces on-and the measurement of nanometer-level displacements of-optically trapped objects. We review progress in the development of optical trapping apparatus, including instrument design considerations, position detection schemes and calibration techniques, with an emphasis on recent advances. We conclude with a brief summary of innovative optical trapping configurations and applications.

    View details for DOI 10.1063/1.1785844

    View details for Web of Science ID 000224754800001

    View details for PubMedID 16878180

  • Forward and reverse motion of single RecBCD molecules on DNA BIOPHYSICAL JOURNAL Perkins, T. T., Li, H. W., Dalal, R. V., Gelles, J., Block, S. M. 2004; 86 (3): 1640-1648

    Abstract

    RecBCD is a processive, DNA-based motor enzyme with both helicase and nuclease activities. We used high-resolution optical trapping to study individual RecBCD molecules moving against applied forces up to 8 pN. Fine-scale motion was smooth down to a detection limit of 2 nm, implying a unitary step size below six basepairs (bp). Episodes of constant-velocity motion over hundreds to thousands of basepairs were punctuated by abrupt switches to a different speed or by spontaneous pauses of mean length 3 s. RecBCD occasionally reversed direction, sliding backward along DNA. Backsliding could be halted by reducing the force, after which forward motion sometimes resumed, often after a delay. Elasticity measurements showed that the DNA substrate was partially denatured during backsliding events, but reannealed concomitant with the resumption of forward movement. Our observations show that RecBCD-DNA complexes can exist in multiple, functionally distinct states that persist for many catalytic turnovers: such states may help tune enzyme activity for various biological functions.

    View details for Web of Science ID 000189377400033

    View details for PubMedID 14990491

  • Kinesin moves by an asymmetric hand-over-hand mechanism SCIENCE Asbury, C. L., Fehr, A. N., Block, S. M. 2003; 302 (5653): 2130-2134

    Abstract

    Kinesin is a double-headed motor protein that moves along microtubules in 8-nanometer steps. Two broad classes of model have been invoked to explain kinesin movement: hand-over-hand and inchworm. In hand-over-hand models, the heads exchange leading and trailing roles with every step, whereas no such exchange is postulated for inchworm models, where one head always leads. By measuring the stepwise motion of individual enzymes, we find that some kinesin molecules exhibit a marked alternation in the dwell times between sequential steps, causing these motors to "limp" along the microtubule. Limping implies that kinesin molecules strictly alternate between two different conformations as they step, indicative of an asymmetric, hand-over-hand mechanism.

    View details for DOI 10.1126/science.1092985

    View details for Web of Science ID 000187385200047

    View details for PubMedID 14657506

  • Backtracking by single RNA polymerase molecules observed at near-base-pair resolution NATURE Shaevitz, J. W., Abbondanzieri, E. A., Landick, R., Block, S. M. 2003; 426 (6967): 684-687

    Abstract

    Escherichia coli RNA polymerase (RNAP) synthesizes RNA with remarkable fidelity in vivo. Its low error rate may be achieved by means of a 'proofreading' mechanism comprised of two sequential events. The first event (backtracking) involves a transcriptionally upstream motion of RNAP through several base pairs, which carries the 3' end of the nascent RNA transcript away from the enzyme active site. The second event (endonucleolytic cleavage) occurs after a variable delay and results in the scission and release of the most recently incorporated ribonucleotides, freeing up the active site. Here, by combining ultrastable optical trapping apparatus with a novel two-bead assay to monitor transcriptional elongation with near-base-pair precision, we observed backtracking and recovery by single molecules of RNAP. Backtracking events ( approximately 5 bp) occurred infrequently at locations throughout the DNA template and were associated with pauses lasting 20 s to >30 min. Inosine triphosphate increased the frequency of backtracking pauses, whereas the accessory proteins GreA and GreB, which stimulate the cleavage of nascent RNA, decreased the duration of such pauses.

    View details for DOI 10.1038/nature02191

    View details for Web of Science ID 000187132800047

    View details for PubMedID 14634670

  • Ubiquitous transcriptional pausing is independent of RNA polymerase backtracking CELL Neuman, K. C., Abbondanzieri, E. A., Landick, R., Gelles, J., Block, S. M. 2003; 115 (4): 437-447

    Abstract

    RNA polymerase (RNAP) transcribes DNA discontinuously, with periods of rapid nucleotide addition punctuated by frequent pauses. We investigated the mechanism of transcription by measuring the effect of both hindering and assisting forces on the translocation of single Escherichia coli transcription elongation complexes, using an optical trapping apparatus that allows for the detection of pauses as short as one second. We found that the vast majority of pauses are brief (1-6 s at 21 degrees C, 1 mM NTPs), and that the probability of pausing at any particular position on a DNA template is low and fairly constant. Neither the probability nor the duration of these ubiquitous pauses was affected by hindering or assisting loads, establishing that they do not result from the backtracking of RNAP along the DNA template. We propose instead that they are caused by a structural rearrangement within the enzyme.

    View details for Web of Science ID 000186627100010

    View details for PubMedID 14622598

  • Sequence-dependent pausing of single lambda exonuclease molecules SCIENCE Perkins, T. T., Dalal, R. V., Mitsis, P. G., Block, S. M. 2003; 301 (5641): 1914-1918

    Abstract

    Lambda exonuclease processively degrades one strand of duplex DNA, moving 5'-to-3' in an ATP-independent fashion. When examined at the single-molecule level, the speeds of digestion were nearly constant at 4 nanometers per second (12 nucleotides per second), interspersed with pauses of variable duration. Long pauses, occurring at stereotypical locations, were strand-specific and sequence-dependent. Pause duration and probability varied widely. The strongest pause, GGCGAT TCT, was identified by gel electrophoresis. Correlating single-molecule dwell positions with sequence independently identified the motif GGCGA. This sequence is found in the left lambda cohesive end, where exonuclease inhibition may contribute to the reduced recombination efficiency at that end.

    View details for DOI 10.1126/science.1088047

    View details for Web of Science ID 000185536700053

    View details for PubMedID 12947034

  • Stepping and stretching - How kinesin uses internal strain to walk processively JOURNAL OF BIOLOGICAL CHEMISTRY Rosenfeld, S. S., Fordyce, P. M., Jefferson, G. M., King, P. H., Block, S. M. 2003; 278 (20): 18550-18556

    Abstract

    The ability of kinesin to travel long distances on its microtubule track without dissociating has led to a variety of models to explain how this remarkable degree of processivity is maintained. All of these require that the two motor domains remain enzymatically "out of phase," a behavior that would ensure that, at any given time, one motor is strongly attached to the microtubule. The maintenance of this coordination over many mechanochemical cycles has never been explained, because key steps in the cycle could not be directly observed. We have addressed this issue by applying several novel spectroscopic approaches to monitor motor dissociation, phosphate release, and nucleotide binding during processive movement by a dimeric kinesin construct. Our data argue that the major effect of the internal strain generated when both motor domains of kinesin bind the microtubule is to block ATP from binding to the leading motor. This effect guarantees the two motor domains remain out of phase for many mechanochemical cycles and provides an efficient and adaptable mechanism for the maintenance of processive movement.

    View details for DOI 10.1074/jbc.M300849200

    View details for Web of Science ID 000182838300126

    View details for PubMedID 12626516

  • Probing the kinesin reaction cycle with a 2D optical force clamp PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Block, S. M., Asbury, C. L., Shaevitz, J. W., Lang, M. J. 2003; 100 (5): 2351-2356

    Abstract

    With every step it takes, the kinesin motor undergoes a mechanochemical reaction cycle that includes the hydrolysis of one ATP molecule, ADPP(i) release, plus an unknown number of additional transitions. Kinesin velocity depends on both the magnitude and the direction of the applied load. Using specialized apparatus, we subjected single kinesin molecules to forces in differing directions. Sideways and forward loads up to 8 pN exert only a weak effect, whereas comparable forces applied in the backward direction lead to stall. This strong directional bias suggests that the primary working stroke is closely aligned with the microtubule axis. Sideways loads slow the motor asymmetrically, but only at higher ATP levels, revealing the presence of additional, load-dependent transitions late in the cycle. Fluctuation analysis shows that the cycle contains at least four transitions, and confirms that hydrolysis remains tightly coupled to stepping. Together, our findings pose challenges for models of kinesin motion.

    View details for DOI 10.1073/pnas.0436709100

    View details for Web of Science ID 000181365000036

    View details for PubMedID 12591957

  • Resource Letter: LBOT-1: Laser-based optical tweezers. American journal of physics Lang, M. J., Block, S. M. 2003; 71 (3): 201-215

    Abstract

    This Resource Letter provides a guide to the literature on optical tweezers, also known as laser-based, gradient-force optical traps. Journal articles and books are cited for the following main topics: general papers on optical tweezers, trapping instrument design, optical detection methods, optical trapping theory, mechanical measurements, single molecule studies, and sections on biological motors, cellular measurements and additional applications of optical tweezers.

    View details for PubMedID 16971965

  • Combined optical trapping and single-molecule fluorescence. Journal of biology Lang, M. J., Fordyce, P. M., Block, S. M. 2003; 2 (1): 6-?

    Abstract

    Two of the mainstay techniques in single-molecule research are optical trapping and single-molecule fluorescence. Previous attempts to combine these techniques in a single experiment - and on a single macromolecule of interest - have met with little success, because the light intensity within an optical trap is more than ten orders of magnitude greater than the light emitted by a single fluorophore. Instead, the two techniques have been employed sequentially, or spatially separated by distances of several micrometers within the sample, imposing experimental restrictions that limit the utility of the combined method. Here, we report the development of an instrument capable of true, simultaneous, spatially coincident optical trapping and single-molecule fluorescence.We demonstrate the capability of the apparatus by studying force-induced strand separation of a rhodamine-labeled, 15 base-pair segment of double-stranded DNA, with force applied perpendicular to the axis of the DNA molecule. As expected, we observed abrupt mechanical transitions corresponding to the unzipping of DNA at a critical force. Transitions occurred concomitant with changes in the fluorescence of dyes attached at the duplex ends, which became unquenched upon strand separation.Through careful optical design, the use of high-performance spectral notch filters, a judicious choice of fluorophores, and the rapid acquisition of data gained by computer-automating the experiment, it is possible to perform combined optical trapping and single-molecule fluorescence. This opens the door to many types of experiment that employ optical traps to supply controlled external loads while fluorescent molecules report concurrent information about macromolecular structure.

    View details for PubMedID 12733997

  • A not-so-cheap stunt SCIENCE Block, S. M. 2002; 297 (5582): 769-769

    View details for Web of Science ID 000177192800019

    View details for PubMedID 12162317

  • An automated two-dimensional optical force clamp for single molecule studies BIOPHYSICAL JOURNAL Lang, M. J., Asbury, C. L., Shaevitz, J. W., Block, S. M. 2002; 83 (1): 491-501

    Abstract

    We constructed a next-generation optical trapping instrument to study the motility of single motor proteins, such as kinesin moving along a microtubule. The instrument can be operated as a two-dimensional force clamp, applying loads of fixed magnitude and direction to motor-coated microscopic beads moving in vitro. Flexibility and automation in experimental design are achieved by computer control of both the trap position, via acousto-optic deflectors, and the sample position, using a three-dimensional piezo stage. Each measurement is preceded by an initialization sequence, which includes adjustment of bead height relative to the coverslip using a variant of optical force microscopy (to +/-4 nm), a two-dimensional raster scan to calibrate position detector response, and adjustment of bead lateral position relative to the microtubule substrate (to +/-3 nm). During motor-driven movement, both the trap and stage are moved dynamically to apply constant force while keeping the trapped bead within the calibrated range of the detector. We present details of force clamp operation and preliminary data showing kinesin motor movement subject to diagonal and forward loads.

    View details for Web of Science ID 000176445800042

    View details for PubMedID 12080136

  • The importance of lattice defects in katanin-mediated microtubule severing in vitro BIOPHYSICAL JOURNAL Davis, L. J., Odde, D. J., Block, S. M., GROSS, S. P. 2002; 82 (6): 2916-2927

    Abstract

    The microtubule-severing enzyme katanin uses ATP hydrolysis to disrupt noncovalent bonds between tubulin dimers within the microtubule lattice. Although its microtubule severing activity is likely important for fundamental processes including mitosis and axonal outgrowth, its mechanism of action is poorly understood. To better understand this activity, an in vitro assay was developed to enable the real-time observation of katanin-mediated severing of individual, mechanically unconstrained microtubules. To interpret the experimental observations, a number of theoretical models were developed and compared quantitatively to the experimental data via Monte Carlo simulation. Models that assumed that katanin acts on a uniform microtubule lattice were incompatible with the in vitro data, whereas a model that assumed that katanin acts preferentially on spatially infrequent microtubule lattice defects was found to correctly predict the experimentally observed breaking rates, number and spatial frequency of severing events, final levels of severing, and sensitivity to katanin concentration over the range 6-300 nM. As a result of our analysis, we propose that defects in the microtubule lattice, which are known to exist but previously not known to have any biological function, serve as sites for katanin activity.

    View details for Web of Science ID 000175802700008

    View details for PubMedID 12023214

  • Coordination of opposite-polarity microtubule motors JOURNAL OF CELL BIOLOGY GROSS, S. P., Welte, M. A., Block, S. M., Wieschaus, E. F. 2002; 156 (4): 715-724

    Abstract

    Many cargoes move bidirectionally, frequently reversing course between plus- and minus-end microtubule travel. For such cargoes, the extent and importance of interactions between the opposite-polarity motors is unknown. In this paper we test whether opposite-polarity motors on lipid droplets in Drosophila embryos are coordinated and avoid interfering with each other's activity, or whether they engage in a tug of war. To this end we impaired the minus-end transport machinery using dynein and dynactin mutations, and then investigated whether plus-end motion was improved or disrupted. We observe a surprisingly severe impairment of plus-end motion due to these alterations of minus-end motor activity. These observations are consistent with a coordination hypothesis, but cannot be easily explained with a tug of war model. Our measurements indicate that dynactin plays a crucial role in the coordination of plus- and minus-end-directed motors. Specifically, we propose that dynactin enables dynein to participate efficiently in bidirectional transport, increasing its ability to stay "on" during minus-end motion and keeping it "off" during plus-end motion.

    View details for DOI 10.1083/jcb.200109047

    View details for Web of Science ID 000176425900013

    View details for PubMedID 11854311

  • Force production by single kinesin motors NATURE CELL BIOLOGY Schnitzer, M. J., Visscher, K., Block, S. M. 2000; 2 (10): 718-723

    Abstract

    Motor proteins such as kinesin, myosin and polymerase convert chemical energy into work through a cycle that involves nucleotide hydrolysis. Kinetic rates in the cycle that depend upon load identify transitions at which structural changes, such as power strokes or diffusive motions, are likely to occur. Here we show, by modelling data obtained with a molecular force clamp, that kinesin mechanochemistry can be characterized by a mechanism in which a load-dependent isomerization follows ATP binding. This model quantitatively accounts for velocity data over a wide range of loads and ATP levels, and indicates that movement may be accomplished through two sequential 4-nm substeps. Similar considerations account for kinesin processivity, which is found to obey a load-dependent Michaelis-Menten relationship.

    View details for Web of Science ID 000089697000017

    View details for PubMedID 11025662

  • Stretching of single collapsed DNA molecules BIOPHYSICAL JOURNAL Baumann, C. G., Bloomfield, V. A., Smith, S. B., Bustamante, C., Wang, M. D., Block, S. M. 2000; 78 (4): 1965-1978

    Abstract

    The elastic response of single plasmid and lambda phage DNA molecules was probed using optical tweezers at concentrations of trivalent cations that provoked DNA condensation in bulk. For uncondensed plasmids, the persistence length, P, decreased with increasing spermidine concentration before reaching a limiting value 40 nm. When condensed plasmids were stretched, two types of behavior were observed: a stick-release pattern and a plateau at approximately 20 pN. These behaviors are attributed to unpacking from a condensed structure, such as coiled DNA. Similarly, condensing concentrations of hexaammine cobalt(III) (CoHex) and spermidine induced extensive changes in the low and high force elasticity of lambda DNA. The high force (5-15 pN) entropic elasticity showed worm-like chain (WLC) behavior, with P two- to fivefold lower than in low monovalent salt. At lower forces, a 14-pN plateau abruptly appeared. This corresponds to an intramolecular attraction of 0.083-0.33 kT/bp, consistent with osmotic stress measurements in bulk condensed DNA. The intramolecular attractive force with CoHex is larger than with spermidine, consistent with the greater efficiency with which CoHex condenses DNA in bulk. The transition from WLC behavior to condensation occurs at an extension about 85% of the contour length, permitting looping and nucleation of condensation. Approximately half as many base pairs are required to nucleate collapse in a stretched chain when CoHex is the condensing agent.

    View details for Web of Science ID 000086349500028

    View details for PubMedID 10733975

  • Dynein-mediated cargo transport in vivo: A switch controls travel distance JOURNAL OF CELL BIOLOGY GROSS, S. P., Welte, M. A., Block, S. M., Wieschaus, E. F. 2000; 148 (5): 945-955

    Abstract

    Cytoplasmic dynein is a microtubule-based motor with diverse cellular roles. Here, we use mutations in the dynein heavy chain gene to impair the motor's function, and employ biophysical measurements to demonstrate that cytoplasmic dynein is responsible for the minus end motion of bidirectionally moving lipid droplets in early Drosophila embryos. This analysis yields an estimate for the force that a single cytoplasmic dynein exerts in vivo (1.1 pN). It also allows us to quantitate dynein-mediated cargo motion in vivo, providing a framework for investigating how dynein's activity is controlled. We identify three distinct travel states whose general features also characterize plus end motion. These states are preserved in different developmental stages. We had previously provided evidence that for each travel direction, single droplets are moved by multiple motors of the same type (Welte et al. 1998). Droplet travel distances (runs) are much shorter than expected for multiple motors based on in vitro estimates of cytoplasmic dynein processivity. Therefore, we propose the existence of a process that ends runs before the motors fall off the microtubules. We find that this process acts with a constant probability per unit distance, and is typically coupled to a switch in travel direction. A process with similar properties governs plus end motion, and its regulation controls the net direction of transport.

    View details for Web of Science ID 000085775700012

    View details for PubMedID 10704445

  • Characterization of photodamage to Escherichia coli in optical traps BIOPHYSICAL JOURNAL Neuman, K. C., Chadd, E. H., Liou, G. F., Bergman, K., Block, S. M. 1999; 77 (5): 2856-2863

    Abstract

    Optical tweezers (infrared laser-based optical traps) have emerged as a powerful tool in molecular and cell biology. However, their usefulness has been limited, particularly in vivo, by the potential for damage to specimens resulting from the trapping laser. Relatively little is known about the origin of this phenomenon. Here we employed a wavelength-tunable optical trap in which the microscope objective transmission was fully characterized throughout the near infrared, in conjunction with a sensitive, rotating bacterial cell assay. Single cells of Escherichia coli were tethered to a glass coverslip by means of a single flagellum: such cells rotate at rates proportional to their transmembrane proton potential (Manson et al.,1980. J. Mol. Biol. 138:541-561). Monitoring the rotation rates of cells subjected to laser illumination permits a rapid and quantitative measure of their metabolic state. Employing this assay, we characterized photodamage throughout the near-infrared region favored for optical trapping (790-1064 nm). The action spectrum for photodamage exhibits minima at 830 and 970 nm, and maxima at 870 and 930 nm. Damage was reduced to background levels under anaerobic conditions, implicating oxygen in the photodamage pathway. The intensity dependence for photodamage was linear, supporting a single-photon process. These findings may help guide the selection of lasers and experimental protocols best suited for optical trapping work.

    View details for Web of Science ID 000083554900047

    View details for PubMedID 10545383

  • Single kinesin molecules studied with a molecular force clamp NATURE Visscher, K., Schnitzer, M. J., Block, S. M. 1999; 400 (6740): 184-189

    Abstract

    Kinesin is a two-headed, ATP-driven motor protein that moves processively along microtubules in discrete steps of 8 nm, probably by advancing each of its heads alternately in sequence. Molecular details of how the chemical energy stored in ATP is coupled to mechanical displacement remain obscure. To shed light on this question, a force clamp was constructed, based on a feedback-driven optical trap capable of maintaining constant loads on single kinesin motors. The instrument provides unprecedented resolution of molecular motion and permits mechanochemical studies under controlled external loads. Analysis of records of kinesin motion under variable ATP concentrations and loads revealed several new features. First, kinesin stepping appears to be tightly coupled to ATP hydrolysis over a wide range of forces, with a single hydrolysis per 8-nm mechanical advance. Second, the kinesin stall force depends on the ATP concentration. Third, increased loads reduce the maximum velocity as expected, but also raise the apparent Michaelis-Menten constant. The kinesin cycle therefore contains at least one load-dependent transition affecting the rate at which ATP molecules bind and subsequently commit to hydrolysis. It is likely that at least one other load-dependent rate exists, affecting turnover number. Together, these findings will necessitate revisions to our understanding of how kinesin motors function.

    View details for Web of Science ID 000081324900059

    View details for PubMedID 10408448

  • Estimating the persistence length of a worm-like chain molecule from force-extension measurements BIOPHYSICAL JOURNAL Bouchiat, C., Wang, M. D., Allemand, J. F., Strick, T., Block, S. M., Croquette, V. 1999; 76 (1): 409-413

    Abstract

    We describe a simple computation of the worm-like chain model and obtain the corresponding force-versus-extension curve. We propose an improvement to the Marko and Siggia interpolation formula of Bustamante et al (Science 1994, 265:1599-1600) that is useful for fitting experimental data. We apply it to the experimental elasticity curve of single DNA molecules. Finally, we present a tool to study the agreement between the worm-like chain model and experiments.

    View details for Web of Science ID 000077870700035

    View details for PubMedID 9876152

  • Force and velocity measured for single molecules of RNA polymerase SCIENCE Wang, M. D., Schnitzer, M. J., Yin, H., Landick, R., Gelles, J., Block, S. M. 1998; 282 (5390): 902-907

    Abstract

    RNA polymerase (RNAP) moves along DNA while carrying out transcription, acting as a molecular motor. Transcriptional velocities for single molecules of Escherichia coli RNAP were measured as progressively larger forces were applied by a feedback-controlled optical trap. The shapes of RNAP force-velocity curves are distinct from those of the motor enzymes myosin or kinesin, and indicate that biochemical steps limiting transcription rates at low loads do not generate movement. Modeling the data suggests that high loads may halt RNAP by promoting a structural change which moves all or part of the enzyme backwards through a comparatively large distance, corresponding to 5 to 10 base pairs. This contrasts with previous models that assumed force acts directly upon a single-base translocation step.

    View details for Web of Science ID 000076727300038

    View details for PubMedID 9794753

  • Kinesin: What gives? CELL Block, S. M. 1998; 93 (1): 5-8

    View details for Web of Science ID 000072910800002

    View details for PubMedID 9546384

  • Leading the procession: new insights into kinesin motors. journal of cell biology Block, S. M. 1998; 140 (6): 1281-1284

    View details for PubMedID 9508762

  • Developmental regulation of vesicle transport in Drosophila embryos: Forces and kinetics CELL Welte, M. A., GROSS, S. P., Postner, M., Block, S. M., Wieschaus, E. F. 1998; 92 (4): 547-557

    Abstract

    In Drosophila embryos, microtubules oriented along apical-basal directions support saltatory vesicle movement. Vesicle traffic includes lipid droplets whose distribution shifts twice during early embryogenesis. Using microscopy, optical tweezers, and a novel squashed-mount embryo preparation, we tracked single droplets and measured the forces these generated. Droplet stalling forces change developmentally, in a roughly quantized fashion, consistent with variation in the number of active motors. We characterized a mutation, klarsicht, that affects droplet transport. Klar+ facilitates changes in force, possibly by coordinating the activity of multiple motors. Alterations in transport affected motion in both apical and basal directions, indicating tight coupling between motors of opposite polarity. Mutations in klar also affect nuclear migration during eye development, suggesting multiple roles for klar-based transport.

    View details for Web of Science ID 000072251500014

    View details for PubMedID 9491895

  • Versatile optical traps with feedback control MOLECULAR MOTORS AND THE CYTOSKELETON, PT B Visscher, K., Block, S. M. 1998; 298: 460-489

    View details for Web of Science ID 000076246300038

    View details for PubMedID 9751903

  • Kinesin hydrolyses one ATP per 8-nm step NATURE Schnitzer, M. J., Block, S. M. 1997; 388 (6640): 386-390

    Abstract

    Kinesin is a two-headed, ATP-dependent motor protein that moves along microtubules in discrete steps of 8 nm. In vitro, single molecules produce processive movement; motors typically take approximately 100 steps before releasing from a microtubule. A central question relates to mechanochemical coupling in this enzyme: how many molecules of ATP are consumed per step? For the actomyosin system, experimental approaches to this issue have generated considerable controversy. Here we take advantage of the processivity of kinesin to determine the coupling ratio without recourse to direct measurements of ATPase activity, which are subject to large experimental uncertainties. Beads carrying single molecules of kinesin moving on microtubules were tracked with high spatial and temporal resolution by interferometry. Statistical analysis of the intervals between steps at limiting ATP, and studies of fluctuations in motor speed as a function of ATP concentration, allow the coupling ratio to be determined. At near-zero load, kinesin molecules hydrolyse a single ATP molecule per 8-nm advance. This finding excludes various one-to-many and many-to-one coupling schemes, analogous to those advanced for myosin, and places severe constraints on models for movement.

    View details for Web of Science ID A1997XM52800053

    View details for PubMedID 9237757

  • Real engines of creation NATURE Block, S. M. 1997; 386 (6622): 217-219

    View details for Web of Science ID A1997WP00300019

    View details for PubMedID 9069274

  • Stretching DNA with optical tweezers BIOPHYSICAL JOURNAL Wang, M. D., Yin, H., Landick, R., Gelles, J., Block, S. M. 1997; 72 (3): 1335-1346

    Abstract

    Force-extension (F-x) relationships were measured for single molecules of DNA under a variety of buffer conditions, using an optical trapping interferometer modified to incorporate feedback control. One end of a single DNA molecule was fixed to a coverglass surface by means of a stalled RNA polymerase complex. The other end was linked to a microscopic bead, which was captured and held in an optical trap. The DNA was subsequently stretched by moving the coverglass with respect to the trap using a piezo-driven stage, while the position of the bead was recorded at nanometer-scale resolution. An electronic feedback circuit was activated to prevent bead movement beyond a preset clamping point by modulating the light intensity, altering the trap stiffness dynamically. This arrangement permits rapid determination of the F-x relationship for individual DNA molecules as short as -1 micron with unprecedented accuracy, subjected to both low (approximately 0.1 pN) and high (approximately 50 pN) loads: complete data sets are acquired in under a minute. Experimental F-x relationships were fit over much of their range by entropic elasticity theories based on worm-like chain models. Fits yielded a persistence length, Lp, of approximately 47 nm in a buffer containing 10 mM Na1. Multivalent cations, such as Mg2+ or spermidine 3+, reduced Lp to approximately 40 nm. Although multivalent ions shield most of the negative charges on the DNA backbone, they did not further reduce Lp significantly, suggesting that the intrinsic persistence length remains close to 40 nm. An elasticity theory incorporating both enthalpic and entropic contributions to stiffness fit the experimental results extremely well throughout the full range of extensions and returned an elastic modulus of approximately 1100 pN.

    View details for Web of Science ID A1997WJ82400034

    View details for PubMedID 9138579

  • Do's and don'ts of poster presentation BIOPHYSICAL JOURNAL Block, S. M. 1996; 71 (6): 3527-3529

    View details for Web of Science ID A1996VX96900062

    View details for PubMedID 9091059

  • Fifty ways to love your lever: Myosin motors CELL Block, S. M. 1996; 87 (2): 151-157

    View details for Web of Science ID A1996VN40300002

    View details for PubMedID 8861898

  • TRANSCRIPTION AGAINST AN APPLIED FORCE SCIENCE Yin, H., Wang, M. D., Svoboda, K., Landick, R., Block, S. M., Gelles, J. 1995; 270 (5242): 1653-1657

    Abstract

    The force produced by a single molecule of Escherichia coli RNA polymerase during transcription was measured optically. Polymerase immobilized on a surface was used to transcribe a DNA template attached to a polystyrene bead 0.5 micrometer in diameter. The bead position was measured by interferometry while a force opposing translocation of the polymerase along the DNA was applied with an optical trap. At saturating nucleoside triphosphate concentrations, polymerase molecules stalled reversibly at a mean applied force estimated to be 14 piconewtons. This force is substantially larger than those measured for the cytoskeletal motors kinesin and myosin and exceeds mechanical loads that are estimated to oppose transcriptional elongation in vivo. The data are consistent with efficient conversion of the free energy liberated by RNA synthesis into mechanical work.

    View details for Web of Science ID A1995TJ29300045

    View details for PubMedID 7502073

  • MACROMOLECULAR PHYSIOLOGY - ONE SMALL STEP FOR MYOSIN NATURE Block, S. M. 1995; 378 (6553): 132-133

    View details for Web of Science ID A1995TD75900029

    View details for PubMedID 7477314

  • Analysis of high resolution recordings of motor movement. Biophysical journal Block, S. M., Svoboda, K. 1995; 68 (4): 230S-239S

    Abstract

    The development of in vitro motility assays for motor proteins has been accompanied by a parallel development of advanced optical instrumentation capable of recording motion at the molecular level. Devices now exist that can record displacements to better than 0.1 nm at bandwidths in excess of 10 kHz, and that can place controlled forces up to many pN on single motors. Ultra-high resolution data from experiments are now pouring in. The analysis and subsequent interpretation of experimental records, which are inevitably contaminated with high levels of thermal noise, remain an ongoing challenge. This essay examines selected issues relating to this challenge and discusses some alternative approaches.

    View details for PubMedID 7787083

  • Fluctuation analysis of kinesin movement. Biophysical journal Svoboda, K., Mitra, P. P., Block, S. M. 1995; 68 (4): 69S-?

    View details for PubMedID 7787104

  • NANOMETERS AND PICONEWTONS - THE MACROMOLECULAR MECHANICS OF KINESIN TRENDS IN CELL BIOLOGY Block, S. M. 1995; 5 (4): 169-175

    Abstract

    Much of our current understanding of the molecular physiology of kinesin has come from in vitro motility assays: indeed, the discovery of kinesin relied upon such assays. By marrying in vitro assays with novel instruments capable of resolving movements on the molecular scale, it has proved possible to make measurements on single motors. Such key parameters as the step size, stepping force, and force-velocity relationship for kinesin have been determined in this fashion, and should soon contribute to a molecular model for the movement of kinesin.

    View details for Web of Science ID A1995QQ30700008

    View details for PubMedID 14732153

  • FLUCTUATION ANALYSIS OF MOTOR PROTEIN MOVEMENT AND SINGLE ENZYME-KINETICS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Svoboda, K., Mitra, P. P., Block, S. M. 1994; 91 (25): 11782-11786

    Abstract

    We studied fluctuations in the displacement of silica beads driven by single molecules of the motor protein kinesin, moving under low mechanical loads at saturating ATP concentrations. The variance in position was significantly smaller than expected for the case of stepwise movement along a regular lattice of positions with exponentially distributed intervals. The small variance suggests that two or more sequential processes with comparable reaction rates dominate the biochemical cycle. The low value is inconsistent with certain recently proposed thermal ratchet models for motor movement as well as with scenarios where the hydrolysis of a single ATP molecule leads to a cluster of several steps. Fluctuation analysis is a potential powerful tool for studying kinetic behavior whenever the output of a single enzyme can be monitored.

    View details for Web of Science ID A1994PW70800004

    View details for PubMedID 7991536

  • FORCE AND VELOCITY MEASURED FOR SINGLE KINESIN MOLECULES CELL Svoboda, K., Block, S. M. 1994; 77 (5): 773-784

    Abstract

    We measured the force-velocity curves of single kinesin molecules attached to silica beads moving in an in vitro motility assay. Optical trapping interferometry was used to track movement with subnanometer precision and to apply calibrated, pN-sized forces to the beads. Velocity decreased linearly with increasing force, and kinesin molecules moved against applied loads of up to 5-6 pN. Comparison of force-velocity curves at limiting and saturating ATP concentrations suggests that the load-dependent diminution in kinesin velocity may be due to a decrease in the net displacement per molecule of ATP hydrolyzed, not simply to a slowing of the ATP turnover rate; kinesin would therefore appear to be a loosely coupled motor.

    View details for Web of Science ID A1994NQ12300016

    View details for PubMedID 8205624

  • A MUTANT HOOK-ASSOCIATED PROTEIN (HAP3) FACILITATES TORSIONALLY INDUCED TRANSFORMATIONS OF THE FLAGELLAR FILAMENT OF ESCHERICHIA-COLI JOURNAL OF MOLECULAR BIOLOGY Fahrner, K. A., Block, S. M., Krishnaswamy, S., Parkinson, J. S., Berg, H. C. 1994; 238 (2): 173-186

    Abstract

    Two mutants with defects in hook-associated protein 3 (HAP3) were isolated that exhibit impaired swimming only when they interact with a solid surface or a semisolid matrix. Motility and chemotaxis were normal in liquid media, even in media containing viscous agents, but cells failed to swarm in 0.28% agar. Mutants appeared to carry a full complement of flagella of normal configuration and length. However, filaments rotating counterclockwise close to a glass surface transformed from normal to straight, while filaments rotating clockwise transformed from curly to straight. Both transformations propagated from base to tip, as expected if torsionally induced. The mutations mapped to the middle of flgL, to structural gene for HAP3, and sequence analysis revealed the same coding change in both mutants: a substitution of cysteine for arginine 168. Our results show that the ability of a filament composed of normal flagellin subunits to resist mechanical stress depends on the structure of the protein (HAP3) to which it is attached at its base. The N-terminal sequence of HAP3 was found to be similar to the N-terminal sequence of flagellin, and the possibility that it provides a nucleation site for the C-terminal region of flagellin is discussed.

    View details for Web of Science ID A1994NH63200005

    View details for PubMedID 8158647

  • BIOLOGICAL APPLICATIONS OF OPTICAL FORCES ANNUAL REVIEW OF BIOPHYSICS AND BIOMOLECULAR STRUCTURE Svoboda, K., Block, S. M. 1994; 23: 247-285

    View details for Web of Science ID A1994NT36300010

    View details for PubMedID 7919782

  • DIRECT OBSERVATION OF KINESIN STEPPING BY OPTICAL TRAPPING INTERFEROMETRY NATURE Svoboda, K., Schmidt, C. F., Schnapp, B. J., Block, S. M. 1993; 365 (6448): 721-727

    Abstract

    Do biological motors move with regular steps? To address this question, we constructed instrumentation with the spatial and temporal sensitivity to resolve movement on a molecular scale. We deposited silica beads carrying single molecules of the motor protein kinesin on microtubules using optical tweezers and analysed their motion under controlled loads by interferometry. We find that kinesin moves with 8-nm steps.

    View details for Web of Science ID A1993MC81200052

    View details for PubMedID 8413650

  • MAKING LIGHT WORK WITH OPTICAL TWEEZERS NATURE Block, S. M. 1992; 360 (6403): 493-495

    Abstract

    Microscopic objects, including biological material, can be remotely manipulated with tightly focused beams of infrared laser light. The use of optical traps, or 'optical tweezers', holds great promise for noninvasive micromanipulation and mechanical measurement in cell biology. Optical tweezers are the 'tractor beams' of today's technology.

    View details for Web of Science ID A1992KA79700071

    View details for PubMedID 1448176

  • CONFORMATION AND ELASTICITY OF THE ISOLATED RED-BLOOD-CELL MEMBRANE SKELETON BIOPHYSICAL JOURNAL Svoboda, K., Schmidt, C. F., Branton, D., Block, S. M. 1992; 63 (3): 784-793

    Abstract

    We studied the structure and elasticity of membrane skeletons from human red blood cells (RBCs) during and after extraction of RBC ghosts with nonionic detergent. Optical tweezers were used to suspend individual cells inside a flow chamber, away from all surfaces; this procedure allowed complete exchange of medium while the low-contrast protein network of the skeleton was observed by high resolution, video-enhanced differential interference-contrast (DIC) microscopy. Immediately following extraction in a 5 mM salt buffer, skeletons assumed expanded, nearly spherical shapes that were uncorrelated with the shapes of their parent RBCs. Judging by the extent of thermal undulations and by their deformability in small flow fields, the bending rigidity of skeletons was markedly lower than that of either RBCs or ghosts. No further changes were apparent in skeletons maintained in this buffer for up to 40 min at low temperatures (T less than 10 degrees C), but skeletons shrank when the ionic strength of the buffer was increased. When the salt concentration was raised to 1.5 M, shrinkage remained reversible for approximately 1 min but thereafter became irreversible. When maintained in 1.5 M salt buffer for longer periods, skeletons continued to shrink, lost flexibility, and assumed irregular shapes: this rigidification was irreversible. At this stage, skeletons closely resembled those isolated in standard bulk preparations. We propose that the transformation to the rigid, irreversibly shrunken state is a consequence of spectrin dimer-dimer reconnections and that these structural rearrangements are thermally activated. We also measured the salt-dependent size of fresh and bulk extracted skeletons. Our measurements suggest that, in situ, the spectrin tethers are flexible, with a persistence length of approximately 10 nm at 150 mM salt.

    View details for Web of Science ID A1992JP02100019

    View details for PubMedID 1420914

  • MORPHOLOGY AND DYNAMICS OF PROTRUDING SPIROCHETE PERIPLASMIC FLAGELLA JOURNAL OF BACTERIOLOGY Charon, N. W., Goldstein, S. F., Block, S. M., CURCI, K., Ruby, J. D., Kreiling, J. A., Limberger, R. J. 1992; 174 (3): 832-840

    Abstract

    We recently characterized the three-dimensional shape of Treponema phagedenis periplasmic flagella (PFs). In the course of these studies, we observed protrusions on swimming cells that resembled PFs. Here we present a detailed characterization of the shape, structure, and motion of these protrusions. Although protrusion formation occurred primarily in wild-type cells during the stationary phase, a large fraction of exponential-phase cells of cell cylinder helicity mutants (greater than 90% of mutant T-52) had protrusions. These results suggest that cells bearing protrusions can still participate in cell division. T. phagedenis protrusions had the identical helix handedness, pitch, and diameter to those of purified PFs. Protrusions were not present on mutants unable to synthesize PFs, but were present in all motile revertants which regained PFs. These results, taken together with electron microscope observations, suggest that protrusions consist of PFs surrounded by an outer membrane sheath. To analyze protrusion movements, we held cells against a coverglass surface with optical tweezers and observed the motion of protrusions by video-enhanced differential interference contrast light microscopy. Protrusions were found to gyrate in both clockwise and counterclockwise directions, and direct evidence was obtained that protrusions rotate. Protrusions were also observed on Treponema denticola and Borrelia burgdorferi. These were also left-handed and had the same helix handedness, pitch, and diameter as purified PFs from their respective species. The PFs from T. denticola had a helix diameter of 0.26 microns and a helix pitch of 0.78 micron; PFs from B. burgdorferi had a helix diameter of 0.28 micron and a helix pitch of 1.48 microns. Protrusions from these spirochete species had similar structures and motion to those of T. phagedenis. Our results present direct evidence that PFs rotate and support previously proposed models of spirochete motility.

    View details for Web of Science ID A1992HE45900022

    View details for PubMedID 1732217

  • VISUALIZATION OF BACTERIAL FLAGELLA BY VIDEO-ENHANCED LIGHT-MICROSCOPY JOURNAL OF BACTERIOLOGY Block, S. M., Fahrner, K. A., Berg, H. C. 1991; 173 (2): 933-936

    Abstract

    We have imaged individual flagellar filaments of Escherichia coli, a motile Streptococcus sp., and Rhizobium meliloti by video-enhanced differential interference-contrast microscopy (Nomarski DIC) and computer-based image processing. This approach has advantages over existing methods in that filaments on living cells can be seen over their entire lengths.

    View details for Web of Science ID A1991ET44600068

    View details for PubMedID 1987174

  • COMPLIANCE OF BACTERIAL POLYHOOKS MEASURED WITH OPTICAL TWEEZERS CYTOMETRY Block, S. M., Blair, D. F., Berg, H. C. 1991; 12 (6): 492-496

    Abstract

    In earlier work, a single-beam gradient force optical trap ("optical tweezers") was used to measure the torsional compliance of flagella in wild-type cells of Escherichia coli that had been tethered to glass by a single flagellum. This compliance was nonlinear, exhibiting a torsionally soft phase up to 180 degrees, followed by a torsionally rigid phase for larger angles. Values for the torsional spring constant in the soft phase were substantially less than estimates based on the rigidity determined for isolated flagellar filaments. It was suggested that the soft phase might correspond to wind-up of the flagellar hook, and the rigid phase to wind-up of the stiffer filament. Here, we have measured the torsional compliance of flagella on cells of an E. coli strain that produces abnormally long hooks but no filaments. The small-angle compliance of these cells, as determined from the elastic rebound of the cell body after wind-up and release, was found to be the same as for wild-type cells. This confirms that the small-angle compliance of wild-type cells is dominated by the response of the hook. Hook flexibility is likely to play a useful role in stabilizing the flagellar bundle.

    View details for Web of Science ID A1991GA50400004

    View details for PubMedID 1764974

  • BEAD MOVEMENT BY SINGLE KINESIN MOLECULES STUDIED WITH OPTICAL TWEEZERS NATURE Block, S. M., Goldstein, L. S., Schnapp, B. J. 1990; 348 (6299): 348-352

    Abstract

    Kinesin, a mechanoenzyme that couples ATP hydrolysis to movement along microtubules, is thought to power vesicle transport and other forms of microtubule-based motility. Here, microscopic silica beads were precoated with carrier protein, exposed to low concentrations of kinesin, and individually manipulated with a single-beam gradient-force optical particle trap ('optical tweezers') directly onto microtubules. Optical tweezers greatly improved the efficiency of the bead assay, particularly at the lowest kinesin concentrations (corresponding to approximately 1 molecule per bead). Beads incubated with excess kinesin moved smoothly along a microtubule for many micrometres, but beads carrying from 0.17-3 kinesin molecules per bead, moved, on average, only about 1.4 microns and then spontaneously released from the microtubule. Application of the optical trap directly behind such moving beads often pulled them off the microtubule and back into the centre of the trap. This did not occur when a bead was bound by an AMP.PNP-induced rigor linkage, or when beads were propelled by several kinesin molecules. Our results are consistent with a model in which kinesin detaches briefly from the microtubule during a part of each mechanochemical cycle, rather than a model in which kinesin remains bound at all times.

    View details for Web of Science ID A1990EJ95500066

    View details for PubMedID 2174512

  • ACTIN CORES OF HAIR-CELL STEREOCILIA SUPPORT MYOSIN MOTILITY PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Shepherd, G. M., Corey, D. P., Block, S. M. 1990; 87 (21): 8627-8631

    Abstract

    The actin cores of hair-cell stereocilia were tested as a substrate for the movement of myosin-coated beads in an in vitro assay. Large numbers of stereocilia from bullfrog sacculi and semicircular canals were isolated by blotting onto coverglasses and were demembranated to expose the polar actin tracks of their cytoskeletal cores. Silica or polystyrene beads, coated with thick filaments of chicken skeletal muscle myosin, were added to this core preparation in the presence of ATP. Myosin-coated beads could reach some of the cores by diffusion alone, but the efficiency and precision of the assay were improved considerably by the use of "optical tweezers" (a gradient-force optical trap) to deposit the beads directly on the cores. Beads applied in this fashion bound and moved unidirectionally at 1-2 microns/s, escaping the retarding force of the trap. Actin filaments within the stereocilia are cross-linked by fimbrin, but this did not appear to interfere with the motility of myosin. Beads coated with optic-lobe kinesin were also tested for movement; these bound and moved unidirectionally at 0.1-0.2 microns/s when applied to microtubule-based kinociliary cores, but not when applied to actin-based stereociliary cores. Our results are consistent with, and lend support to, a model for hair cell adaptation in which a molecular motor such as myosin maintains tension on the mechanically gated transduction channels. Optical tweezers and video-enhanced differential interference contrast optics provide high efficiency and improved optical resolution for the in vitro analysis of myosin motility.

    View details for Web of Science ID A1990EG22000091

    View details for PubMedID 2236074

  • COMPLIANCE OF BACTERIAL FLAGELLA MEASURED WITH OPTICAL TWEEZERS NATURE Block, S. M., Blair, D. F., Berg, H. C. 1989; 338 (6215): 514-518

    Abstract

    The development of the gradient force optical particle trap ('optical tweezers') has made it possible to manipulate biological materials using a single beam of laser light. Optical traps can produce forces in the microdyne range on intact cells without causing overt damage: such forces are sufficient to arrest actively swimming bacteria and can overcome torque generated by the flagellar motor of a bacterium tethered to a glass surface by a flagellar filament. By calibrating the trapping force against Stokes' drag and measuring the twist that is sustained by this force, we determined the torsional compliance of flagella in tethered Escherichia coli and a motile Streptococcus. Flagella behaved as linear torsion springs for roughly half a revolution, but became much more rigid when turned beyond this point in either direction.

    View details for Web of Science ID A1989U008400059

    View details for PubMedID 2648159

  • MOVEMENT OF MYOSIN FRAGMENTS INVITRO - DOMAINS INVOLVED IN FORCE PRODUCTION CELL Hynes, T. R., Block, S. M., White, B. T., Spudich, J. A. 1987; 48 (6): 953-963

    Abstract

    We have used the Nitella-based movement assay to localize the site of force production in myosin. Methods were developed to use nonfilamentous myosin or proteolytic fragments of myosin in place of the thick filaments used in the original assay. In the experiments described here, the tail of myosin or its subfragments is anchored via antibodies to the surface of small particles. Nonfilamentous myosin or its subfragments move along Nitella actin cables at speeds similar to those obtained with filamentous myosin. We generated short HMM, a myosin fragment containing the heads and only 400 A of the tail. Although short HMM lacks the "hinge" region proposed by Harrington to be the site of force generation, and is incapable of forming thick filaments, it moves along actin at speeds above 1 micron/sec. Therefore, neither a thick filament nor the carboxy-terminal 1100 A of the tail is required for movement along actin. The results indicate that force production occurs in or near the myosin heads.

    View details for Web of Science ID A1987G694600006

    View details for PubMedID 3548997

  • TEMPORAL COMPARISONS IN BACTERIAL CHEMOTAXIS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Segall, J. E., Block, S. M., Berg, H. C. 1986; 83 (23): 8987-8991

    Abstract

    Responses of tethered cells of Escherichia coli to impulse, step, exponential-ramp or exponentiated sine-wave stimuli are internally consistent, provided that allowance is made for the nonlinear effect of thresholds. This result confirms that wild-type cells exposed to stimuli in the physiological range make short-term temporal comparisons extending 4 sec into the past: the past second is given a positive weighting, the previous 3 sec are given a negative weighting, and the cells respond to the difference. cheRcheB mutants (defective in methylation and demethylation) weight the past second in a manner similar to the wild type, but they do not make short-term temporal comparisons. When exposed to small steps delivered iontophoretically, they fail to adapt over periods of up to 12 sec; when exposed to longer steps in a flow cell, they partially adapt, but with a decay time of greater than 30 sec. cheZ mutants use a weighting that extends at least 40 sec into the past. The gain of the chemotactic system is large: the change in occupancy of one receptor molecule produces a significant response.

    View details for Web of Science ID A1986F147100036

    View details for PubMedID 3024160

  • MYOSIN MOVEMENT INVITRO - A QUANTITATIVE ASSAY USING ORIENTED ACTIN CABLES FROM NITELLA METHODS IN ENZYMOLOGY Sheetz, M. P., Block, S. M., Spudich, J. A. 1986; 134: 531-544

    View details for Web of Science ID A1986H889700050

    View details for PubMedID 3821577

  • A miniature flow cell designed for rapid exchange of media under high-power microscope objectives. Journal of general microbiology Berg, H. C., Block, S. M. 1984; 130 (11): 2915-2920

    Abstract

    The design, fabrication and use of a flow cell that allows rapid displacement of media viewed by short working distance, high power objectives are described. The cell has a small internal depth (about 0.04 cm), small volume (about 0.05 ml), and is chemically inert. It has been tested extensively in studies of tethered bacteria.

    View details for PubMedID 6396378

  • SUCCESSIVE INCORPORATION OF FORCE-GENERATING UNITS IN THE BACTERIAL ROTARY MOTOR NATURE Block, S. M., Berg, H. C. 1984; 309 (5967): 470-472

    Abstract

    Mot mutants of Escherichia coli are paralysed: their flagella appear to be intact but do not rotate. The motA and motB gene products are found in the cytoplasmic membrane; they do not co-purify with flagellar basal bodies isolated in neutral detergents. Silverman et al. found that mot mutants could be ' resurrected ' through protein synthesis directed by lambda transducing phages carrying the wild-type genes. Here, we have studied this activation at the level of a single flagellar motor. Cells of a motB strain carrying plasmids in which transcription of the wild-type motB gene was controlled by the lac promoter were tethered to a glass surface by a single flagellum. These cells began to spin within several minutes after the addition of a lac inducer, and their rotational speed changed in a series of equally spaced steps. As many as 7 steps were seen in individual cells and, from the final speeds attained, as many as 16 steps could be inferred. These experiments show that each flagellar motor contains several independent force-generating units comprised, at least in part, of motB protein.

    View details for Web of Science ID A1984ST80800061

    View details for PubMedID 6374467

  • COORDINATION OF FLAGELLA ON FILAMENTOUS CELLS OF ESCHERICHIA-COLI JOURNAL OF BACTERIOLOGY Ishihara, A., Segall, J. E., Block, S. M., Berg, H. C. 1983; 155 (1): 228-237

    Abstract

    Video techniques were used to study the coordination of different flagella on single filamentous cells of Escherichia coli. Filamentous, nonseptate cells were produced by introducing a cell division mutation into a strain that was polyhook but otherwise wild type for chemotaxis. Markers for its flagellar motors (ordinary polyhook cells that had been fixed with glutaraldehyde) were attached with antihook antibodies. The markers were driven alternately clockwise and counterclockwise, at angular velocities comparable to those observed when wild-type cells are tethered to glass. The directions of rotation of different markers on the same cell were not correlated; reversals of the flagellar motors occurred asynchronously. The bias of the motors (the fraction of time spent spinning counterclockwise) changed with time. Variations in bias were correlated, provided that the motors were within a few micrometers of one another. Thus, although the directions of rotation of flagellar motors are not controlled by a common intracellular signal, their biases are. This signal appears to have a limited range.

    View details for Web of Science ID A1983QX62000031

    View details for PubMedID 6345503

  • ADAPTATION KINETICS IN BACTERIAL CHEMOTAXIS JOURNAL OF BACTERIOLOGY Block, S. M., Segall, J. E., Berg, H. C. 1983; 154 (1): 312-323

    Abstract

    Cells of Escherichia coli, tethered to glass by a single flagellum, were subjected to constant flow of a medium containing the attractant alpha-methyl-DL-aspartate. The concentration of this chemical was varied with a programmable mixing apparatus over a range spanning the dissociation constant of the chemoreceptor at rates comparable to those experienced by cells swimming in spatial gradients. When an exponentially increasing ramp was turned on (a ramp that increases the chemoreceptor occupancy linearly), the rotational bias of the cells (the fraction of time spent spinning counterclockwise) changed rapidly to a higher stable level, which persisted for the duration of the ramp. The change in bias increased with ramp rate, i.e., with the time rate of change of chemoreceptor occupancy. This behavior can be accounted for by a model for adaptation involving proportional control, in which the flagellar motors respond to an error signal proportional to the difference between the current occupancy and the occupancy averaged over the recent past. Distributions of clockwise and counterclockwise rotation intervals were found to be exponential. This result cannot be explained by a response regular model in which transitions between rotational states are generated by threshold crossings of a regular subject to statistical fluctuation; this mechanism generates distributions with far too many long events. However, the data can be fit by a model in which transitions between rotational states are governed by first-order rate constants. The error signal acts as a bias regulator, controlling the values of these constants.

    View details for Web of Science ID A1983QK26000039

    View details for PubMedID 6339475

  • LIGHT AND DARK-ADAPTATION IN PHYCOMYCES LIGHT-GROWTH RESPONSE JOURNAL OF GENERAL PHYSIOLOGY Lipson, E. D., Block, S. M. 1983; 81 (6): 845-859

    Abstract

    Sporangiophores of the fungus Phycomyces exhibit adaptation to light stimuli over a dynamic range of 10(10). This range applies to both phototropism and the closely related light-growth response; in the latter response, the elongation rate is modulated transiently by changes in the light intensity. We have performed light- and dark-adaptation experiments on growing sporangiophores using an automated tracking machine that allows a continuous measurement of growth velocity under controlled conditions. The results are examined in terms of the adaptation model of Delbrück and Reichardt (1956, Cellular Mechanisms in Differentiation and Growth, 3-44). The "level of adaptation," A, was inferred from responses to test pulses of light by means of a series of intensity-response curves. For dark adaptation to steps down in the normal intensity range (10(-6)-10(-2) W/m2), A decays exponentially with a time constant b = 6.1 +/- 0.3 min. This result is in agreement with the model. Higher-order kinetics are indicated, however, for dark adaptation in the high-intensity range (10(-2)-1 W/m2). Adaptation in this range is compared with predictions of a model relating changes in A to the inactivation and recovery of a receptor pigment. In response to steps up in intensity in the normal range, A was found to increase rapidly, overshoot the applied intensity level, and then relax to that level within 40 min. These results are incompatible with the Delbrück-Reichardt model or any simple generalizations of it. The asymmetry and overshoot are similar to adaptation phenomena observed in systems as diverse as bacterial chemotaxis and human vision. It appears likely that light and dark adaptation in Phycomyces are mediated by altogether different processes.

    View details for Web of Science ID A1983QV60300005

    View details for PubMedID 6875507

  • IMPULSE RESPONSES IN BACTERIAL CHEMOTAXIS CELL Block, S. M., Segall, J. E., Berg, H. C. 1982; 31 (1): 215-226

    Abstract

    The chemotactic behavior of Escherichia coli has been studied by exposing cells tethered by a single flagellum to pulses of chemicals delivered iontophoretically. Normally, wild-type cells spin alternately clockwise and counterclockwise, changing their direction on the average approximately once per second. When cells were exposed to a very brief diffusive wave of attractant, the probability of spinning counterclockwise quickly peaked, then fell below the prestimulus value, returning to baseline within a few seconds; repellent responses were similar but inverted. The width of the response indicates that cells integrate sensory inputs over a period of seconds, while the biphasic character implies that they also take time derivatives of these inputs. The sensory system is maximally tuned to concentration changes that occur over a span of approximately 2 sec, an interval over which changes normally occur when cells swim in spatial gradients; it is optimized to extract information from signals subject to statistical fluctuation. Impulse responses of cells defective in methylation were similar to those of wild-type cells, but did not fall as far below the baseline, indicating a partial defect in adaptation. Impulse responses of cheZ mutants were aberrant, indicating a serious defect in excitation.

    View details for Web of Science ID A1982PQ46100024

    View details for PubMedID 6760985

Conference Proceedings


  • Molecule by molecule, the physics and chemistry of life: SMB 2007. Block, S. M., Larson, M. H., Greenleaf, W. J., Herbert, K. M., Guydosh, N. R., Anthony, P. C. 2007: 193-197

    Abstract

    Interdisciplinary work in the life sciences at the boundaries of biology, chemistry and physics is making enormous strides. This progress was showcased at the recent Single Molecule Biophysics conference.

    View details for PubMedID 17372599

  • Role of ponticulin in pseudopod dynamics, cell-cell adhesion, and mechanical stability of an amoeboid membrane skeleton LUNA, E. J., HITT, A. L., Shutt, D., Wessels, D., Soll, D., Jay, P., Hug, C., Elson, E. L., Vesley, A., Downey, G. P., Wang, M., Block, S. M., Sigurdson, W., Sachs, F. MARINE BIOLOGICAL LABORATORY. 1998: 345-346

    View details for Web of Science ID 000074759100022

    View details for PubMedID 11536879

  • Statistical kinetics of processive enzymes Schnitzer, M. J., Block, S. M. COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT. 1995: 793-802

    View details for Web of Science ID A1995VA12500084

    View details for PubMedID 8824454

  • BIOPHYSICAL PRINCIPLES OF SENSORY TRANSDUCTION Block, S. M. ROCKEFELLER UNIV PRESS. 1992: 1-17

    View details for Web of Science ID A1992BW27V00001

    View details for PubMedID 1369757

Stanford Medicine Resources: