Biomechanics, One Molecule at a Time (1999)

Single-molecule observation has come of age. Parallel developments of sensitive mechanical probes and single fluorophore detection now fuse into unique combinations, allowing investigators to examine shape and chemical transitions of single molecules with ever increasing precision and finesse. Optical trapping, using focused laser beams to constrain dielectric particles in solution (for reviews see Ref. 1$$Google Scholar), has emerged as a widely used and versatile tool to examine mechanically interesting proteins and DNA. The associated forces of light on matter can be rendered sufficiently weak that single molecules compete with them. In most applications to date, molecules of interest are attached to uniform dielectric beads, which are trapped and used as handles to configure an appropriate experimental geometry. One can detect bead position with high precision, monitoring biological activity by tracking probe displacement. Such methods allow accurate, quantitative characterization of force and displacement transients driven or experienced by single molecules, providing a unique edge in deciphering the underlying mechanisms and reaction schemes. Several biomolecules have met variants on this theme. Here, we focus attention on three classes: processive motors, nonprocessive motors, and proteins experiencing significant strain. A processive enzyme undergoes multiple productive catalytic cycles per diffusional encounter with its binding partner. Illustrating this, a processive motor protein binds its polymer track and advances along it through several unitary cycles before dissociating (for reviews, see Refs. 2Howard J. Nature. 1997; 389: 561-567Crossref PubMed Scopus (458) Google Scholar and 3Lohman T.M. Thorn K. Vale R.D. Cell. 1998; 93: 9-12Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Two widely studied examples are kinesin, involved in vesicle transport, and RNA polymerase (RNAP), 1The abbreviations used are: RNAP, RNA polymerase; pN, piconewton(s); AFM, atomic force microscope. involved in DNA transcription. Under in vitro conditions with purified proteins, a single kinesin molecule can move along its microtubule track for several microns before dissociating (4Howard J. Hudspeth A.J. Vale R.D. Nature. 1989; 342: 154-158Crossref PubMed Scopus (733) Google Scholar, 5Block S.M. Goldstein L.S. Schnapp B.J. Nature. 1990; 348: 348-352Crossref PubMed Scopus (838) Google Scholar, 6Vale R.D. Funatsu T. Pierce D.W. Romberg L. Harada Y. Yanagida T. Nature. 1996; 380: 451-453Crossref PubMed Scopus (608) Google Scholar). To further characterize this movement, Svoboda et al. (7Svoboda K. Schmidt C.F. Schnapp B.J. Block S.M. Nature. 1993; 365: 721-727Crossref PubMed Scopus (1563) Google Scholar) attached kinesin at low density to silica beads, captured such a bead with an optical trap, and moved it to close proximity of microtubules fixed on a microscope coverslip (Fig. 1a). They observed discrete advances between dwell positions spaced 8 nm apart (Fig. 1b). Kinesin continued to advance the optically trapped and thus elastically loaded bead until it no longer could, a point at which resistive load is termed the "stall force," measured at 5 pN (7Svoboda K. Schmidt C.F. Schnapp B.J. Block S.M. Nature. 1993; 365: 721-727Crossref PubMed Scopus (1563) Google Scholar) to 7 pN (8Kojima H. Muto E. Higuchi H. Yanagida T. Biophys. J. 1997; 73: 2012-2022Abstract Full Text PDF PubMed Scopus (257) Google Scholar). The bead eventually detached and fell back to the trap center before kinesin rebound the microtubule and began again to pull the bead. These pioneering experiments uncovered the step character and distance but left open the question of correspondence between ATP turnover and the 8-nm advances. To address this, others have approached the kinetic scheme underlying mechanical activity by examining the distribution of dwell periods between unitary advances. Unfortunately, fast stepping or noisy data preclude unambiguous identification of all such transitions. Investigators circumvented this by examining the staircase-like data records at the ensemble level, examining the mean and variance of bead position across the ensemble as a function of time after the records begin (9Svoboda K. Mitra P.P. Block S.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11782-11786Crossref PubMed Scopus (264) Google Scholar, 10Schnitzer M.J. Block S.M. Nature. 1997; 388: 386-390Crossref PubMed Scopus (627) Google Scholar). The ensemble average should increase with time, the slope being the average kinesin velocity. The ensemble variance, however, contains information regarding the dwell period distribution. If kinesin stepping proceeds with clocklike regularity, meaning a large number of processes are comparably rate-limiting for each mechanical step, then all kinesin encounters with its track should proceed with the same time course. The variance of bead position across the data record ensemble should remain constant, equaling zero if the records are synchronized. If instead the stepping events are stochastic, individual data records need not follow the same course and the ensemble variance will increase with time. As Svoboda et al. (9Svoboda K. Mitra P.P. Block S.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11782-11786Crossref PubMed Scopus (264) Google Scholar) calculate, if a single chemical transition limits the rate of each mechanical step, the variance rises with a slope of velocity times step size. If two kinetically comparable and rate-limiting chemical transitions precede each mechanical step, the variance rises with half that slope. If two mechanical steps follow each rate-limiting chemical transition, the variance rises with twice that slope. Svoboda et al. (9Svoboda K. Mitra P.P. Block S.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11782-11786Crossref PubMed Scopus (264) Google Scholar) used such measurements to demonstrate that under saturating ATP conditions, each mechanical advance is rate-limited by two kinetically comparable processes. Schnitzeret al. (10Schnitzer M.J. Block S.M. Nature. 1997; 388: 386-390Crossref PubMed Scopus (627) Google Scholar) performed like experiments under a broad range of ATP concentrations and showed ensemble variance under limited ATP was consistent with a single rate-limiting process, ATP binding, per mechanical advance. This demonstrates that single ATP binding events separate all or nearly all 8-nm advances at all ATP concentrations. Kojima et al. (8Kojima H. Muto E. Higuchi H. Yanagida T. Biophys. J. 1997; 73: 2012-2022Abstract Full Text PDF PubMed Scopus (257) Google Scholar) and Hua et al.(11Hua W. Young E.C. Fleming M.L. Gelles J. Nature. 1997; 388: 390-393Crossref PubMed Scopus (278) Google Scholar) have reached similar conclusions by fitting the distribution of dwell times separating detected step transitions. A similar experimental geometry has been used to examine the behavior of kinesin under load either along or against its direction of movement (12Coppin C.M. Pierce D.W. Hsu L. Vale R.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8539-8544Crossref PubMed Scopus (213) Google Scholar). Visscher et al. (13Visscher K. Block S.M. Methods Enzymol. 1998; 298: 460-489Crossref PubMed Scopus (125) Google Scholar) developed an instrument capable of maintaining a fixed trap-bead separation and thus fixed system tension. One expects that single-molecule experiments using such a technique should clarify the nature of chemomechanical coupling as a function of load, shedding more light on observed slippage and stalling phenomenon. Despite the relative maturity of single-molecule kinesin experiments, more surprises seem likely. Investigators continue to puzzle over how so small a molecule with no visible means of 8–16-nm extension can behave as the above step measurements suggest (for review, see Ref. 14Block S.M. Cell. 1998; 93: 5-8Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). In contrast to kinesin, RNAP does not strike one as a molecular motor, as its biological function involves DNA transcription as opposed to moving cargo or generating tension. However, RNA synthesis requires using free energy released from nucleotide condensation for generating force to advance along the DNA template. Because RNAP usually does not dissociate from its track before finishing the transcript, it is a processive molecular motor (for review, see Ref. 15Gelles J. Landick R. Cell. 1998; 93: 13-16Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). RNAP must thread through and presumably rotate a helical DNA strand, precluding the scheme of Svoboda et al. (7Svoboda K. Schmidt C.F. Schnapp B.J. Block S.M. Nature. 1993; 365: 721-727Crossref PubMed Scopus (1563) Google Scholar), fixing the motor upon a trapped bead and the polymer track upon the surface. Instead, Yin et al. (16Yin H. Wang M.D. Svoboda K. Landick R. Block S.M. Gelles J. Science. 1995; 270: 1653-1657Crossref PubMed Scopus (468) Google Scholar) adapted an experiment designed by Shafer et al. (17Schafer D.A. Gelles J. Sheetz M.P. Landick R. Nature. 1991; 352: 444-448Crossref PubMed Scopus (232) Google Scholar), in which a surface-mounted RNAP binds and pulls on a solution DNA duplex, attached to a bead on its transcriptionally downstream end. As RNAP advances upon its template, the bead is drawn closer to the surface. Shafer et al. (17Schafer D.A. Gelles J. Sheetz M.P. Landick R. Nature. 1991; 352: 444-448Crossref PubMed Scopus (232) Google Scholar) monitored this single transcription reaction by observing the diffusive range of the tethered bead as a function of time. Yin et al.(16Yin H. Wang M.D. Svoboda K. Landick R. Block S.M. Gelles J. Science. 1995; 270: 1653-1657Crossref PubMed Scopus (468) Google Scholar) extended this experiment by trapping the bead. In early work, Yin et al. (16Yin H. Wang M.D. Svoboda K. Landick R. Block S.M. Gelles J. Science. 1995; 270: 1653-1657Crossref PubMed Scopus (468) Google Scholar) measured the "stall force" against which the molecule ceased movement, analogous to that described above for kinesin. At high optical trap strength, they observed many transcribing RNAP molecules to stall against loads of 12.3 ± 3.5 pN, likely an underestimate because some complexes did not stall and some likely suffered damage from laser light exposure. Wang et al. (18Wang M.D. Schnitzer M.J. Yin H. Landick R. Gelles J. Block S.M. Science. 1998; 282: 902-907Crossref PubMed Scopus (731) Google Scholar) used a feedback scheme to increase effective trap strength while reducing light exposure; they measured stall forces of 21–27 pN, considerably above the 5–7 pN required to stall single kinesin motors. RNAP may need such high forces to untangle DNA secondary structure during transcription. Once the feedback system was turned off following stall, RNAP recovered its transcription activity only after a 0–30-s delay. Wang et al. (18Wang M.D. Schnitzer M.J. Yin H. Landick R. Gelles J. Block S.M. Science. 1998; 282: 902-907Crossref PubMed Scopus (731) Google Scholar) observed intermittent stalls for comparable times when the enzyme moved against low load, suggesting these times reflect transition rates out of a nonproductive state, a phenomenon with no analogue in the kinesin data records. Finally, Wang et al. (19Wang M.D. Yin H. Landick R. Gelles J. Block S.M. Biophys. J. 1997; 72: 1335-1346Abstract Full Text PDF PubMed Scopus (1125) Google Scholar) corrected for DNA and other elasticity as well as geometry to estimate protein movement along DNA from detected bead displacement (18Wang M.D. Schnitzer M.J. Yin H. Landick R. Gelles J. Block S.M. Science. 1998; 282: 902-907Crossref PubMed Scopus (731) Google Scholar). They computed RNAP velocity as a function of resistive force, a measurement of central importance in elucidating the reaction steps underlying movement for this and other motor enzymes (20Svoboda K. Block S.M. Cell. 1994; 77: 773-784Abstract Full Text PDF PubMed Scopus (700) Google Scholar, 21Hunt A.J. Gittes F. Howard J. Biophys. J. 1994; 67: 766-781Abstract Full Text PDF PubMed Scopus (286) Google Scholar). The load dependence of velocity provides information regarding enzyme behavior in the absence of load. Wanget al. (18Wang M.D. Schnitzer M.J. Yin H. Landick R. Gelles J. Block S.M. Science. 1998; 282: 902-907Crossref PubMed Scopus (731) Google Scholar) showed velocity remained fairly constant against variable load until it fell off sharply just below the stall force, suggesting that the rate-limiting process under low load does not involve movement along the DNA long axis. From fitting the sharp velocity drop under loads approaching stall, Wang et al.(18Wang M.D. Schnitzer M.J. Yin H. Landick R. Gelles J. Block S.M. Science. 1998; 282: 902-907Crossref PubMed Scopus (731) Google Scholar) estimated that the force affects the underlying unitary RNAP reaction cycle over a distance spanning at least 5–10 base pairs. This may indicate that optical load induces stall through a conformational strain 5–10 times larger than the distance traversed in a given unitary cycle. Alternatively, load may engage a more complex pathway, perhaps favoring an inactive state that follows slippage by 5–10 base pairs in the transcriptionally upstream direction. Transient pauses in observed RNAP movement records lend further support to the long suggested existence of such a state. One expects future experiments of like character to examine the influence of specific DNA sequences or protein cofactors on RNAP activity. More precise position detectors will track the enzyme with better than single base pair resolution. Moreover, once the problem of photoinduced enzyme damage is solved and instrumental drift is suitably reduced, the study of slow RNAP movement under limiting nucleotide conditions should allow step and chemomechanical coupling measurements analogous to those described for kinesin. Through these and other experiments, RNAP seems likely to usher in single-molecule study of a broad array of DNA-based proteins. A non-processive enzyme undergoes at most one productive catalytic cycle per diffusional encounter with its binding partner (for reviews, see Refs. 2Howard J. Nature. 1997; 389: 561-567Crossref PubMed Scopus (458) Google Scholar and 22Spudich J.A. Nature. 1994; 372: 515-518Crossref PubMed Scopus (421) Google Scholar). Although one or a few molecules of non-processive motors cannot transport cargo over long distances, large ensembles of them can move along their tracks at faster rates than comparable ensembles of processive ones. Several members of the myosin superfamily, motors that act on the filamentous polymer actin, can be so classified. Not many but only one unitary advance follows each binding event, demanding a different experimental geometry to capture this advance in detectable probe motion. Simple attachment of binding partners to bead and surface, respectively, would not suffice, because small protein movement would likely cause bead rotation, which is not detectable. Using a dual beam optical trap (23Simmons R.M. Finer J.T. Chu S. Spudich J.A. Biophys. J. 1996; 70: 1813-1822Abstract Full Text PDF PubMed Scopus (415) Google Scholar), Finer et al. (24Finer J.T. Simmons R.M. Spudich J.A. Nature. 1994; 368: 113-119Crossref PubMed Scopus (1537) Google Scholar) approached this problem by attaching each end of an actin filament to a polystyrene bead and using the trapped beads as handles. They stretched the filament to tension and moved it near silica spheres, mounted on a coverslip surface and decorated sparsely with skeletal muscle myosin II molecules (Fig. 2a). Myosin bound the filament, often pulled the bead from trap center, and maintained the deflected position for a variable dwell period before releasing (Fig. 2b, top). Because skeletal myosin II detaches from actin only after binding ATP from solution, Finer et al. (24Finer J.T. Simmons R.M. Spudich J.A. Nature. 1994; 368: 113-119Crossref PubMed Scopus (1537) Google Scholar) reduced ATP concentrations to extend the dwell period and clarify bead deflections. Such measured deflections ranged from 10 to 15 nm, more consistent with a tightly coupled mechanical ratchet than some predictions of much larger displacement values (cf. Ref. 25Harada Y. Sakurada K. Aoki T. Thomas D.D. Yanagida T. J. Mol. Biol. 1990; 216: 49-68Crossref PubMed Scopus (449) Google Scholar). This pioneering experiment left open issues regarding probe thermal diffusion, detection of all binding events, protein orientation, myosin mounting on the surface, artifacts of geometry, and compliant linkages in the bead-actin system, all addressed in the work that followed. All optical trap measurements to date indicate a unitary displacement of 5–15 nm (24Finer J.T. Simmons R.M. Spudich J.A. Nature. 1994; 368: 113-119Crossref PubMed Scopus (1537) Google Scholar, 26Molloy J.E. Burns J.E. Kendrick-Jones J. Tregear R.T. White D.C. Nature. 1995; 378: 209-212Crossref PubMed Scopus (520) Google Scholar, 27Guilford W.H. Dupuis D.E. Kennedy G. Wu J. Patlak J.B. Warshaw D.M. Biophys. J. 1997; 72: 1006-1021Abstract Full Text PDF PubMed Scopus (217) Google Scholar, 28Mehta A.D. Finer J.T. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7927-7931Crossref PubMed Scopus (115) Google Scholar, 29Ishijima A. Kojima H. Funatsu T. Tokunaga M. Higuchi H. Tanaka H. Yanagida T. 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J. 1998; 75: Full Text Full Text PDF PubMed Scopus Google Scholar) have used with few motors to displacement as a function of between the long of actin and They bead near 10 nm when the two are 5 nm when the are near zero when the are 5 nm in the same actin filament direction when the proteins are Such indicate that myosin binds and actin in the same by when Moreover, they suggest measurements of oriented myosin may underestimate the unitary step et al. A. Kojima H. Funatsu T. Tokunaga M. Higuchi H. Tanaka H. Yanagida T. Cell. 1998; 92: 161-171Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar) a synthesis of the Finer et al. (24Finer J.T. Simmons R.M. Spudich J.A. Nature. 1994; 368: 113-119Crossref PubMed Scopus (1537) Google Scholar) optical trapping geometry with a technique used by Funatsu et al. T. Harada Y. Tokunaga M. K. Yanagida T. 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PubMed Scopus Google Scholar), when pulled at the same A broad of interesting molecules have study using optical to or them. for such study proteins that force, motors, or those biomolecules that to it in interesting The experiments described above a of which of and of movement and experiments in described will likely upon methods to at once protein shape and the associated binding events and