title: Kinetics and thermodynamics of the rate-limiting conformational change in the actomyosin V mechanochemical cycle.
abstract: We used FRET to examine the kinetics and thermodynamics of structural changes associated with ADP release in myosin V, which is thought to be a strain-sensitive step in many muscle and non-muscle myosins. We also explored essential dynamics using FIRST/FRODA starting with three different myosin V X-ray crystal structures to examine intrinsic flexibility and correlated motions. Our steady-state and time-resolved FRET analysis demonstrates a temperature-dependent reversible conformational change in the nucleotide-binding pocket (NBP). Our kinetic results demonstrate that the NBP goes from a closed to an open conformation prior to the release of ADP, while the actin-binding cleft remains closed. Interestingly, we find that the temperature dependence of the maximum actin-activated myosin V ATPase rate is similar to the pocket opening step, demonstrating that this is the rate-limiting structural transition in the ATPase cycle. Thermodynamic analysis demonstrates that the transition from the open to closed NBP conformation is unfavorable because of a decrease in entropy. The intrinsic flexibility analysis is consistent with conformational entropy playing a role in this transition as the MV.ADP structure is highly flexible compared to the MV.APO structure. Our experimental and modeling studies support the conclusion of a novel post-power-stroke actomyosin.ADP state in which the NBP and actin-binding cleft are closed. The novel state may be important for strain sensitivity as the transition from the closed to open NBP conformation may be altered by lever arm position.
authors.affiliation: Department of Physics and Optical Science, University of North Carolina at Charlotte, 9201 University City Blvd., Charlotte, NC 28223, USA.
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Jacobs
authors.forename: Darshan
authors.initials: D
authors.lastname: Trivedi
authors.forename: Charles
authors.initials: C
authors.lastname: David
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Yengo
mesh_terms: D000205:Actomyosin / Q000737:chemistry* / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D018360:Crystallography, X-Ray; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D024701:Myosin Type V / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013816:Thermodynamics
chemical_list: D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000205:Actomyosin; D024701:Myosin Type V