Cyclic association and dissociation of cross-bridges formed between myosin molecules in the thick filament and actin molecules in the thin filament are coupled to ATP hydrolysis. The coupled cycle can lead to sliding of the filaments thus resulting in generation of force and production of work. An understanding of the mechanism by which chemical free energy released from ATP hydrolysis is converted to mechanical work (contraction) requires detailed knowledge of molecular interactions that are involved in the sliding motions. Evidence points to the presence of an energy transduction """"""""loop"""""""" within the subfragment l(Sl) region of myosin. Intersite communication between biologically relevant sites (actin-binding, ATP) within this loop is an important feature of energy transduction in muscle. The first part of the proposed work addresses the association and dissociation kinetics of actin with fluorescently-labeled Sl. We will examine the kinetics of these reactions with both unregulated and regulated actin filaments by using stopped-flow and temperature-jump relaxation fluorometry. The second project is designed to understand the relationship between intersite communication and the dynamic properties of the heavy chain of Sl. We will determine molecular distances by fluorescence resonance energy transfer (FRET) and use these distances to elucidate structural changes that occur in Sl and the complex formed between actin and Sl resulting from muscle activation.. Intramolecularly crosslinked Sl and intermolecularly crosslinked proteins will be used for these studies. The changes in FRET induced by biologically relevant perturbations will be kinetically resolved in rapid kinetic experiments. Both kinetic and spectroscopic information will be incorporated into contractile models. Synthetic peptides will be used as models to study the flexibility of a short segment of Sl heavy chain. They will be studied by NMR, fluorescence and computer simulation. Finally, we will obtain collaboratively mutant nSl proteins that have specific sequence alteration for structure/function studies.
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