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.
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