The work described in this proposal seeks to characterize the structural basis of contraction at the molecular level. Although it is well established that muscle contraction, and acto-myosin based motility in non-muscle cells, occurs by a displacement of the actin-attached SFl crossbridge of myosin while it is hydrolyzing MgATP, the structural basis of this displacement is not known. The fact that there is now a body of evidence indicating that the SFl crossbridge is flexible and that one region in its catalytic heavy chain subunit can undergo marked nucleotide induced movements, suggest that the displacement involved in force generation may take place wholly within the SF1 segment of myosin. The proposed work seeks to characterize the structure-function relationships in the SFl segment of the crossbridge especially with respect to its heavy chain (HC) subunit. The goals of this proposal are (i) to examine the tertiary structure of the active HC and the changes it undergoes when the protein binds MgADP and MgATP; (ii) to employ monoclonal antibodies (McAb's) to probe the topography of the native HC and SFl; (iii) to examine the HC contribution to the flexibility exhibited by SFl; (iv) to determine the consequences of alkali light chain removal on the nature of the acto-HC rigor interaction; (v) to determine whether changes in the separation between the SHl and SH2 thiols in SFl are translated into changes elsewhere in the molecule; (vi) to examine the structural basis for the destabilization. To achieve these goals the structure of the active HC will be subjected to detailed probing involving the use of following methodologies - crosslinking to determine sites of proximity; the action of McAb's to sequences directly or indirectly involved with ATPase and actin functions; NMR spectroscopy to examine flexibility; binding and limited proteolysis to examine SFl structure in presence and absence of alkali light chain and, the procedure of thermal ion-exchange to study the stability under physiological conditions. These studies will provide significant information, currently missing, about the structural basis of the contractile function of the myosin molecule with respect to its catalytic heavy chain subunit.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS015319-07
Application #
3396130
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1978-12-01
Project End
1989-12-31
Budget Start
1986-01-01
Budget End
1986-12-31
Support Year
7
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Case Western Reserve University
Department
Type
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
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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