The long range goal of our laboratory is to understand the structural basis for actomyosin contractility and motility and its regulation at the molecular level. The ability of muscles to contract,k cells to under go division and shape changes are fundamental biological processes based in part or whole on the actomyosin interaction which is necessary for any organism to survive. Although much information is known about these processes and their regulatory control, little is known of the structural changes responsible for force generation and movement and its regulation in terms of the molecular structure of myosin or actomyosin. The present proposal seeks to characterize the location and extent of the structural changes which occur in the S1 region of the myosin crossbridge when it interacts with MgATP and actin. This will be done by examining the proximal relationship in S1 in different conformational states associated with the contractile cycle by employing heterofunctional, photoactivatable reagents to modify the protein. This will allow for the characterization of regions which remain static or which move during the contractile cycle. In addition the substructure of S1 will be studied by examining how the protein unfolds and refolds in the presence of nucleotide or actin. This will be done by employing specific modification of certain side-chains of the protein and by the use of antibodies to follow the unfolding and refolding process. This project should provided detailed information about the organization of the structure of S1 and the changes which it undergoes to produce contraction in muscle and actomyosin based motility in non- muscle cells.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
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Physical Biochemistry Study Section (PB)
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Case Western Reserve University
Schools of Engineering
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Gopal, D; Burke, M (1996) Myosin subfragment 1 hydrophobicity changes associated with different nucleotide-induced conformations. Biochemistry 35:506-12
Gopal, D; Bobkov, A A; Schwonek, J P et al. (1995) Structural basis for actomyosin chemomechanical transduction by non-nucleoside triphosphate analogues. Biochemistry 34:12178-84
Gopal, D; Burke, M (1995) Formation of stable inhibitory complexes of myosin subfragment 1 using fluoroscandium anions. J Biol Chem 270:19282-6
Agarwal, R; Rajasekharan, K N; Burke, M (1991) Identification of the site of photocross-linking formed in the absence of magnesium nucleotide from SH2 (Cys-697) in myosin subfragment 1 labeled with 4'-maleimidylbenzophenone. J Biol Chem 266:2272-5
Agarwal, R; Burke, M (1991) Temperature-induced changes in the flexibility of the loop between SH1 (Cys-707) and SH3 (Cys-522) in myosin subfragment 1 detected by cross-linking. Arch Biochem Biophys 290:1-6
Rajasekharan, K N; Morita, J I; Mayadevi, M et al. (1991) Formation and properties of smooth muscle myosin 20-kDa light chain-skeletal muscle myosin hybrids and photocrosslinking from the maleimidylbenzophenone-labeled light chain to the heavy chain. Arch Biochem Biophys 288:584-90
Rajasekharan, K N; Burke, M (1989) Structural changes in myosin subfragment 1 by mild denaturation and proteolysis probed by antibodies. Arch Biochem Biophys 274:304-11
Rajasekharan, K N; Mayadevi, M; Burke, M (1989) Studies of ligand-induced conformational perturbations in myosin subfragment 1. An examination of the environment about the SH2 and SH1 thiols using a photoprobe. J Biol Chem 264:10810-9
Rajasekharan, K N; Burke, M (1989) Trypsinolysis promotes disulfide formation between 21- and 50-kilodalton segments of myosin subfragment 1 during reaction with 5,5'-dithiobis(2-nitrobenzoic acid). Biochemistry 28:6473-7
Zaager, S; Burke, M (1988) Subunit interactions of skeletal muscle myosin and myosin subfragment 1. Evidence for heavy chain-alkali light chain association-dissociation equilibrium. J Biol Chem 263:1513-7

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