title: Myosin conformational states determined by single fluorophore polarization.
abstract: Muscle contraction is powered by the interaction of the molecular motor myosin with actin. With new techniques for single molecule manipulation and fluorescence detection, it is now possible to correlate, within the same molecule and in real time, conformational states and mechanical function of myosin. A spot-confocal microscope, capable of detecting single fluorophore polarization, was developed to measure orientational states in the smooth muscle myosin light chain domain during the process of motion generation. Fluorescently labeled turkey gizzard smooth muscle myosin was prepared by removal of endogenous regulatory light chain and re-addition of the light chain labeled at cysteine-108 with the 6-isomer of iodoacetamidotetramethylrhodamine (6-IATR). Single myosin molecule fluorescence polarization data, obtained in a motility assay, provide direct evidence that the myosin light chain domain adopts at least two orientational states during the cyclic interaction of myosin with actin, a randomly disordered state, most likely associated with myosin whereas weakly bound to actin, and an ordered state in which the light chain domain adopts a finite angular orientation whereas strongly bound after the powerstroke.
authors.affiliation: Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, VT 05405, USA. warshaw@salus.med.uvm.edu
authors.forename: D M
authors.initials: DM
authors.lastname: Warshaw
authors.forename: E
authors.initials: E
authors.lastname: Hayes
authors.forename: D
authors.initials: D
authors.lastname: Gaffney
authors.forename: A M
authors.initials: AM
authors.lastname: Lauzon
authors.forename: J
authors.initials: J
authors.lastname: Wu
authors.forename: G
authors.initials: G
authors.lastname: Kennedy
authors.forename: K
authors.initials: K
authors.lastname: Trybus
authors.forename: S
authors.initials: S
authors.lastname: Lowey
authors.forename: C
authors.initials: C
authors.lastname: Berger
mesh_terms: D000818:Animals; D005453:Fluorescence; D018613:Microscopy, Confocal; D018994:Myosin Light Chains / Q000737:chemistry; D009218:Myosins / Q000737:chemistry*; D011487:Protein Conformation; D014422:Turkeys
chemical_list: D018994:Myosin Light Chains; D009218:Myosins