The proposed experiments are based upon the amino acid sequence studies on actin and myosin that have been carried out in this laboratoy over the past several years. Intermolecular crosslinking of F-actin will be carried out using bifunctional reagents, some of which will be photoactivatible, the actin will be digested, and crosslinked peptides will be isolated. They will then be sequenced and the specific residues that are crosslinked will be localized in the overall sequence of actin; it is anticipated that these results will yield information concerning the distances between specific amino acid sidechains, which will in turn aid in understanding the orientation of actin monomers in F-actin. Studies on myosin will include completion of the amino acid sequence of the heavy chain of skeletal muscle myosin, and comparative studies on cardiac myosin. The latter will be focused upon the methylated lysines, since recent data suggests that, in skeletal muscle, one of the trimethyllysines is near the ATPase site, while the monomethyllysine is found in only a fraction of the chains, and thus may represent a means to regulate or modify the properties of myosin. In collaborative studies, monoclonal antibodies to small peptides from myosin will be used to try to determine, by electron microscopy of tungslen-shadowed antibody-myosin complexes, the location of key residues within the native structure of the myosin head. It is anticipated that this work will yield new insights into the mechanisms of assembly and function of contractile proteins in the heart, other muscles, and the cytoplasm of other cells.
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