Breaking the millisecond barrier: single molecule motors wobble to find their next binding sites.

Over the last 20 years, advances in single molecule biophysics have allowed the structure and dynamics of biological macromolecules to be examined at increasingly high resolution (1xSingle-molecule biophysics: at the interface of biology, physics and chemistry. Deniz, A.A., Mukhopadhyay, S., and Lemke, E.A. J. Roy. Soc. Interface. 2008; 5: 15–45Crossref | PubMed | Scopus (136)See all References1). The study of motor proteins in particular has benefitted tremendously from the development of numerous optical techniques for examining both the mechanical (2xThe biochemical kinetics underlying actin movement generated by one and many skeletal muscle myosin molecules. Baker, J.E., Brosseau, C...., and Warshaw, D.M. Biophys. J. 2002; 82: 2134–2147Abstract | Full Text | Full Text PDF | PubMedSee all References, 3xMyosin V exhibits a high duty cycle and large unitary displacement. Moore, J.R., Krementsova, E.B...., and Warshaw, D.M. J. Cell Biol. 2001; 155: 625–635Crossref | PubMedSee all References) and structural changes (4xMeasurement of single macromolecule orientation by total internal reflection fluorescence polarization microscopy. Forkey, J.N., Quinlan, M.E., and Goldman, Y.E. Biophys. J. 2005; 89: 1261–1271Abstract | Full Text | Full Text PDF | PubMed | Scopus (61)See all References, 5xThree-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization. Forkey, J.N., Quinlan, M.E...., and Goldman, Y.E. Nature. 2003; 422: 399–404Crossref | PubMed | Scopus (287)See all References, 6xPolarized fluorescence microscopy of individual and many kinesin motors bound to axonemal microtubules. Peterman, E.J., Sosa, H...., and Moerner, W.E. Biophys. J. 2001; 81: 2851–2863Abstract | Full Text | Full Text PDF | PubMedSee all References, 7xOrientation of the myosin light chain region by single molecule total internal reflection fluorescence polarization microscopy. Quinlan, M.E., Forkey, J.N., and Goldman, Y.E. Biophys. J. 2005; 89: 1132–1142Abstract | Full Text | Full Text PDF | PubMed | Scopus (36)See all References, 8xADP-induced rocking of the kinesin motor domain revealed by single-molecule fluorescence polarization microscopy. Sosa, H., Peterman, E.J...., and Goldstein, L.S. Nat. Struct. Biol. 2001; 8: 540–544Crossref | PubMed | Scopus (97)See all References, 9xMyosin conformational states determined by single fluorophore polarization. Warshaw, D.M., Hayes, E...., and Berger, C. Proc. Natl. Acad. Sci. USA. 1998; 95: 8034–8039Crossref | PubMed | Scopus (94)See all References) required for biological motility at the molecular level. But as the spatial resolution in single-molecule fluorescence experiments has improved to the nanometer level, this has somewhat been at the expense of temporal resolution, which has remained on the millisecond timescale. While millisecond time resolution is more than adequate for detecting changes in position or orientation between stable conformations of the protein of interest, important dynamic information during the transitions between these states, which occur on the microsecond timescale, are lost. Beausang et al. (10xTilting and wobble of myosin V by high-speed single molecule polarized fluorescence microscopy. Beausang, J.F., Shroder, D.Y...., and Goldman, Y.E. Biophys. J. 2013; 104: 1263–1273Abstract | Full Text | Full Text PDF | PubMed | Scopus (15)See all References10) overcome this temporal limitation in a report in this issue of Biophysical Journal, by observing brief changes in the microsecond wobble of its lever arm as myosin V steps along actin filaments using single-molecule fluorescence polarization in a total internal reflection fluorescence (TIRF) microscopy assay (polTIRF).Unlike improvements in spatial resolution that have relied on the ability to collect more photons, the authors achieve this significant breakthrough in time-resolved structural studies at the single-molecule level by collecting fewer photons. The 50-fold improvement in time resolution relative to previous polTIRF measurements from the same group (4xMeasurement of single macromolecule orientation by total internal reflection fluorescence polarization microscopy. Forkey, J.N., Quinlan, M.E., and Goldman, Y.E. Biophys. J. 2005; 89: 1261–1271Abstract | Full Text | Full Text PDF | PubMed | Scopus (61)See all References, 5xThree-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization. Forkey, J.N., Quinlan, M.E...., and Goldman, Y.E. Nature. 2003; 422: 399–404Crossref | PubMed | Scopus (287)See all References, 7xOrientation of the myosin light chain region by single molecule total internal reflection fluorescence polarization microscopy. Quinlan, M.E., Forkey, J.N., and Goldman, Y.E. Biophys. J. 2005; 89: 1132–1142Abstract | Full Text | Full Text PDF | PubMed | Scopus (36)See all References, 11xProtein structural dynamics by single-molecule fluorescence polarization. Forkey, J.N., Quinlan, M.E., and Goldman, Y.E. Prog. Biophys. Mol. Biol. 2000; 74: 1–35Crossref | PubMed | Scopus (95)See all References) was achieved by being able to switch the polarization of the excitation light every 100 μs instead of 5–10 ms, and by moving to a single-photon counting detection system. However, the improved time-resolution results in an unavoidable reduction in signal/noise that necessitates corresponding improvements in the ability to discern changes in polarization output. The authors solve this problem by implementing an innovative analysis scheme, using a maximum-likelihood, multitrace change-point algorithm previously developed in their lab (12xChange-point analysis for single-molecule polarized total internal reflection fluorescence microscopy experiments. Beausang, J.F., Goldman, Y.E., and Nelson, P.C. Methods Enzymol. 2011; 487: 431–463Crossref | PubMed | Scopus (3)See all References12).Thus, beyond the improvements to the polTIRF instrumentation presented here, Beausang et al. (10xTilting and wobble of myosin V by high-speed single molecule polarized fluorescence microscopy. Beausang, J.F., Shroder, D.Y...., and Goldman, Y.E. Biophys. J. 2013; 104: 1263–1273Abstract | Full Text | Full Text PDF | PubMed | Scopus (15)See all References10) provide a blueprint for future advances in other time-resolved, single-molecule structural studies, involving a combination of enhancements in both the hardware and software components of data acquisition and analysis. In their work, this powerful new approach has resulted in elucidation of a number of new observations about the myosin V stepping mechanism, including what is believed to be the first direct observation of rotational wobble of the myosin V motor domain as it thermally searches for the next available actin binding site (Fig. 1Fig. 1 b) between stable attachments to the actin filament before (Fig. 1Fig. 1 a) and after the step (Fig. 1Fig. 1 c). While this wobble was predicted from earlier optical trap experiments (13xThe gated gait of the processive molecular motor, myosin V. Veigel, C., Wang, F...., and Molloy, J.E. Nat. Cell Biol. 2002; 4: 59–65Crossref | PubMed | Scopus (243)See all References13) and detected from lateral fluctuations of a relatively large gold particle attached to the lever arm (14xDynamics of the unbound head during myosin V processive translocation. Dunn, A.R. and Spudich, J.A. Nat. Struct. Mol. Biol. 2007; 14: 246–248Crossref | PubMed | Scopus (105)See all References14), these experiments used a small organic fluorescent probe to demonstrate the high-microsecond rotational mobility of the lever arm when it is detached from actin. Given the increasing recognition of the importance of thermal fluctuations in macromolecular function, the instrumentation and analysis techniques developed by Beausang et al. (10xTilting and wobble of myosin V by high-speed single molecule polarized fluorescence microscopy. Beausang, J.F., Shroder, D.Y...., and Goldman, Y.E. Biophys. J. 2013; 104: 1263–1273Abstract | Full Text | Full Text PDF | PubMed | Scopus (15)See all References10), extending measurements of single-molecule structural dynamics into the microsecond timescale, open up exciting new avenues of investigation for researchers working with a number of different biological systems.Figure 1Schematic of myosin V stepping along an actin filament. (a) Upon binding ATP the trailing head detaches and (b) undergoes a high mobility thermal search for the next available binding site (the microsecond-scale rotational wobble directly observed in the report by Beausang et al. (10xTilting and wobble of myosin V by high-speed single molecule polarized fluorescence microscopy. Beausang, J.F., Shroder, D.Y...., and Goldman, Y.E. Biophys. J. 2013; 104: 1263–1273Abstract | Full Text | Full Text PDF | PubMed | Scopus (15)See all References10) in this issue of Biophysical Journal), where (c) it binds stably, allowing for product release and the associated powerstroke (adapted from Dunn and Spudich (14xDynamics of the unbound head during myosin V processive translocation. Dunn, A.R. and Spudich, J.A. Nat. Struct. Mol. Biol. 2007; 14: 246–248Crossref | PubMed | Scopus (105)See all References14)).View Large Image | View Hi-Res Image | Download PowerPoint Slide