We propose to continue to explore the structural changes within the myosin and actomyosin systems during ATPase activity, and to relate changes to elementary steps of the reaction mechanisms. This will be done by attaching onto the contractile proteins specific probes which are sensitive to the distances between each other. Unique sites are the nucleotide binding sites of actin and myosin, the alkali light chain thiol groups and cysteine 373 on actin. We will continue to explore other unique sites using the transglutaminase method, as well as developing the nucleotide synthesis of probes in which the probe is located at various known distances from the ribose moiety of the nucleotide. We will continue to explore the actomyosin ATPase mechanism using spectroscopic and 18O-isotopic techniques. Thus we will look for new intermediates and conformational states of the actomyosin system through the use of 19F-NMR and fluorine labelled nucleotides such as 3'-fluoro-3'-deoxy 3'-fluoro-3'-deoxy-xyloadenine 5'-diphosphate. We will complete our studies on the stereochemistry of myosin ATPase reaction by determining which enantiomer of (16O; 17O; 18O) thiophosphate is formed when stereospecific (gamma-S; gamma-18O) ATP is hydrolyzed by 17 O-water in the presence of myosin.