title: ATP turnover by individual myosin molecules hints at two conformers of the myosin active site.
abstract: Coupling of ATP hydrolysis to structural changes in the motor domain is fundamental to the driving of motile functions by myosins. Current understanding of this chemomechanical coupling is primarily based on ensemble average measurements in solution and muscle fibers. Although important, the averaging could potentially mask essential details of the chemomechanical coupling, particularly for mixed populations of molecules. Here, we demonstrate the potential of studying individual myosin molecules, one by one, for unique insights into established systems and to dissect mixed populations of molecules where separation can be particularly challenging. We measured ATP turnover by individual myosin molecules, monitoring appearance and disappearance of fluorescent spots upon binding/dissociation of a fluorescent nucleotide to/from the active site of myosin. Surprisingly, for all myosins tested, we found two populations of fluorescence lifetimes for individual myosin molecules, suggesting that termination of fluorescence occurred by two different paths, unexpected from standard kinetic schemes of myosin ATPase. In addition, molecules of the same myosin isoform showed substantial intermolecular variability in fluorescence lifetimes. From kinetic modeling of our two fluorescence lifetime populations and earlier solution data, we propose two conformers of the active site of myosin, one that allows the complete ATPase cycle and one that dissociates ATP uncleaved. Statistical analysis and Monte Carlo simulations showed that the intermolecular variability in our studies is essentially due to the stochastic behavior of enzyme kinetics and the limited number of ATP binding events detectable from an individual myosin molecule with little room for static variation among individual molecules, previously described for other enzymes.
authors.affiliation: Department of Molecular and Cell Physiology and Department of Biophysical Chemistry, Hannover Medical School, D-30625 Hannover, Germany.
authors.forename: Mamta
authors.initials: M
authors.lastname: Amrute-Nayak
authors.forename: Katharina-Antonia
authors.initials: KA
authors.lastname: Lambeck
authors.forename: Ante
authors.initials: A
authors.lastname: Radocaj
authors.forename: Helen Elisabeth
authors.initials: HE
authors.lastname: Huhnt
authors.forename: Tim
authors.initials: T
authors.lastname: Scholz
authors.forename: Nils
authors.initials: N
authors.lastname: Hahn
authors.forename: Georgios
authors.initials: G
authors.lastname: Tsiavaliaris
authors.forename: Wilhelm J
authors.initials: WJ
authors.lastname: Walter
authors.forename: Bernhard
authors.initials: B
authors.lastname: Brenner
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D003198:Computer Simulation; D006868:Hydrolysis; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D009010:Monte Carlo Method; D009119:Muscle Contraction / Q000502:physiology*; D009218:Myosins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
chemical_list: D020033:Protein Isoforms; D000255:Adenosine Triphosphate; D009218:Myosins
keywords: ATP dwell times; TIRF microscopy; dwell time distribution; single ATP turnover assay