Elucidation of the mechanism of muscle contraction requires further information concerning structure- function relationships in the myosin head (S1), the key regions containing the actin and nucleotide interaction sites. Knowledge of the precise mode of subunit interactions within the myosin molecule as well as the process of assembly of myosin molecules by interaction of their coiled-coil portions into thick filaments is also incomplete. Thus the application is focusing on four areas: (1) The topography of myosin S1 will be explored by (a) establishing proximity relationships in myosin S1 with the use of photoactivatable crosslinkers under various conditions (in the presence of nucleotide, actin, etc.); (b) elucidating the process of polypeptide chain unfolding under partial denaturation conditions such as methanol or heat treatment; (c) studying the effects and locations of antibodies made against specific segments of peptides of S1. (2) The interaction of actin and myosin will be explored by (a) identifying the regions within the interface between actin and S1 after crosslinking under conditions corresponding to strong and weak binding states; (b) using synthetic peptides with sequences corresponding to the putative binding regions of S1 and actin; (c) using actin mutants with thiols introduced at the putative interfaces by site directed mutagenesis. (3) The mechanism of thick filament formation by myosin rod association including parallel and anti-parallel packing will be studied by crosslinking the filaments with the zero length crosslinker 1-ethyl-3- [3- (dimethyl amino) propyl] carbodiimide (EDC) or with thiol specific reagents. New thiols will be introduced by genetic engineering, which would permit the attachment of fluorescent probes or crosslinkers at desired locations. (4) The light chain/ heavy chain interaction will be studied by identifying the regions involved in the crosslinking between heavy chain and alkali light chains with EDC and N-hydroxy succinimide. We will also crosslink light chains to heavy chains with thiol specific photoactivatable reagents using hybrid S1 with light chains from different species or light chains, expressed after site directed mutagenesis. The knowledge of nomal muscle will serve as a reference and may eventually prove useful in the diagnosis, treatment, and prevention of disease involving muscle tissues and the cardiovascular system.
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