During muscular contraction myosin crossbridge and actin filament enter into cyclic interaction which ultimately results in tension development. The energy source for this mechanical work is the free energy released from ATP hydrolysis catalyzed by actomyosin. Most of the free energy drop appears to be associated with substrate binding to myosin. The mechanism by which energy is transduced in muscle is poorly understood. During catalysis and upon binding to actin the myosin structure undergoes structural changes and these changes are believed to play an important role in the overall contractile mechanism. The proposed research will investigate certain structural aspects of skeletal myosin subfragment-1 (S-1) which contains both the substrate and actin binding sites and changes of these structural features induced by nucleotide and actin binding. The presteady state kinetics of the acto-S-1 ATPase will also be investigated. Proximity relationship of S-1 will be investigated by fluorescence energy transfer (FET) and its solution conformation by other fluorescence methods with both extrinsic and intrinsic fluorophores. The role of S-1 heavy chain in myosin function will be investigated by examining the effect of its limited cleavage on the chemical reactivity and structure of S-1, and the dynamic nature of the S-1 active site region. Fluorescence spectroscopic techniques will be used for these studies. The folding of S-1 heavy chain will be studied by using crosslinking reagents with specificities for lysyl and sulfhydryl groups. Finally, the kinetic mechanism for the interaction of S-1 with actin will be elucidated by using stopped flow FET and fluorescence polarization. The presteady state kinetics of S-1 ATPase and acto S-1 ATPase will be investigated by FET with fluorescent substrates. These kinetic studies will be performed with special attention paid to the possible role of the DTNB light chain in the kinetic events.