A primary aim of this work is to extend our understanding of the relationship between the mechanism of nucleoside triphosphate (NTP) hydrolysis and the mechanical properties of muscle fibers with an emphasis upon determining which step or steps of the nucleotide triphosphate hydrolysis mechanism limits the maximum shortening velocity. Investigators have shown that a series of nucleoside triphosphates (NTPS) differ widely in their ability to support shortening and motility as measured in skinned muscle fibers (in collaboration with Drs. Roger Cooke and Ed Pate) and using the `in vitro' motility assay. In the first two years of this proposal the investigators plan to complete the study of the mechanism of hydrolysis of a series of nucleoside triphosphates by actomyosin using stopped-flow fluorescence, light scattering, and rapid chemical quench method. From this work they plan to determine for which steps of the hydrolysis mechanism changes in rate constants alter the mechanical and motile properties of muscle. To accomplish this the investigators will make a detailed comparison of the rate constants of the kinetic mechanism of the actomyosin NTP hydrolysis mechanism with the mechanical properties of muscle fibers and shortening velocity measured with the in vitro motility assay. A parallel approach is to determine the differences in amino acid sequence that are responsible for the differences in enzymatic and contractile activities of different myosins. A seven percent difference in the amino acid sequence between alpha and beta cardiac myosin primarily grouped in four clusters is responsible for the difference in mechanical and enzymatic properties of cardiac myosin isozymes. A similar pattern of more extensive sequence changes produces a three fold difference in the rates of actin activated ATP hydrolysis, a five fold difference in shortening velocity, and a ten fold difference in rate of ADP dissociation from acto-S1 between cardiac and fast skeletal myosin. Recent data (Uyeda et al. 1994) indicate that the rate of actin-activated ATP hydrolysis is dependent upon the sequence of amino acids 624-638, which are located in the junction between the 25 and 50 kDA regions of the myosin molecule. Investigators plan to use a combination of recombinant expression of chimeric myosins (chimerzymes), steady state kinetics, pre-steady state kinetics, and motility assay measurements to determine which amino acid changes are responsible for the observed differences in the kinetic mechanism and functional differences. Understanding which amino acids alter the rate and equilibrium constants of the hydrolysis mechanism and shortening velocity in muscle will provide a better understanding of the molecular mechanism of muscle contraction.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL041776-07
Application #
2519302
Study Section
Biochemistry Study Section (BIO)
Project Start
1988-12-01
Project End
2001-08-31
Budget Start
1997-09-01
Budget End
1998-08-31
Support Year
7
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Eastern Virginia Medical School
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Norfolk
State
VA
Country
United States
Zip Code
23501
Heeley, David H; Belknap, Betty; White, Howard D (2006) Maximal activation of skeletal muscle thin filaments requires both rigor myosin S1 and calcium. J Biol Chem 281:668-76
Heeley, David H; Belknap, Betty; White, Howard D (2002) Mechanism of regulation of phosphate dissociation from actomyosin-ADP-Pi by thin filament proteins. Proc Natl Acad Sci U S A 99:16731-6
Ikebe, R; Reardon, S; Mitsui, T et al. (1999) Role of the N-terminal region of the regulatory light chain in the dephosphorylation of myosin by myosin light chain phosphatase. J Biol Chem 274:30122-6
Kambara, T; Rhodes, T E; Ikebe, R et al. (1999) Functional significance of the conserved residues in the flexible hinge region of the myosin motor domain. J Biol Chem 274:16400-6
White, H D; Belknap, B; Webb, M R (1997) Kinetics of nucleoside triphosphate cleavage and phosphate release steps by associated rabbit skeletal actomyosin, measured using a novel fluorescent probe for phosphate. Biochemistry 36:11828-36
Persechini, A; White, H D; Gansz, K J (1996) Different mechanisms for Ca2+ dissociation from complexes of calmodulin with nitric oxide synthase or myosin light chain kinase. J Biol Chem 271:62-7
Walker, M; Trinick, J; White, H (1995) Millisecond time resolution electron cryo-microscopy of the M-ATP transient kinetic state of the acto-myosin ATPase. Biophys J 68:87S-91S
Walker, M; White, H; Belknap, B et al. (1994) Electron cryomicroscopy of acto-myosin-S1 during steady-state ATP hydrolysis. Biophys J 66:1563-72
Cooke, R; White, H; Pate, E (1994) A model of the release of myosin heads from actin in rapidly contracting muscle fibers. Biophys J 66:778-88
Pate, E; White, H; Cooke, R (1993) Determination of the myosin step size from mechanical and kinetic data. Proc Natl Acad Sci U S A 90:2451-5

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