title: Tilting and wobble of myosin V by high-speed single-molecule polarized fluorescence microscopy.
abstract: Myosin V is biomolecular motor with two actin-binding domains (heads) that take multiple steps along actin by a hand-over-hand mechanism. We used high-speed polarized total internal reflection fluorescence (polTIRF) microscopy to study the structural dynamics of single myosin V molecules that had been labeled with bifunctional rhodamine linked to one of the calmodulins along the lever arm. With the use of time-correlated single-photon counting technology, the temporal resolution of the polTIRF microscope was improved ~50-fold relative to earlier studies, and a maximum-likelihood, multitrace change-point algorithm was used to objectively determine the times when structural changes occurred. Short-lived substeps that displayed an abrupt increase in rotational mobility were detected during stepping, likely corresponding to random thermal fluctuations of the stepping head while it searched for its next actin-binding site. Thus, myosin V harnesses its fluctuating environment to extend its reach. Additional, less frequent angle changes, probably not directly associated with steps, were detected in both leading and trailing heads. The high-speed polTIRF method and change-point analysis may be applicable to single-molecule studies of other biological systems.
authors.affiliation: Department of Physics and Astronomy, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: John F
authors.initials: JF
authors.lastname: Beausang
authors.forename: Deborah Y
authors.initials: DY
authors.lastname: Shroder
authors.forename: Philip C
authors.initials: PC
authors.lastname: Nelson
authors.forename: Yale E
authors.initials: YE
authors.lastname: Goldman
mesh_terms: D000818:Animals; D008856:Microscopy, Fluorescence; D009068:Movement; D024701:Myosin Type V / Q000737:chemistry* / Q000378:metabolism; D012235:Rhodamines / Q000737:chemistry; D012399:Rotation; D013997:Time Factors
chemical_list: D012235:Rhodamines; D024701:Myosin Type V