At the molecular level muscle contraction is driven by the cyclic interaction of two proteins, actin and myosin. Myosin posses two striking functional features that allow it to act as a molecular motor, namely its ability to 1.) generate force and motion with actin filaments through a putative powerstroke and 2.) alter its affinity for actin by more than four orders of magnitude at different stages of its enzymatic MgATPase cycle. This proposal will explicitly examine the structural basis of the powerstroke and alteration in actin binding affinity in myosin using a combination of state-of-the-art molecular biological, spectroscopic, transient kinetic, and mechanical assays. New sites for the specific incorporation of fluorescent probes in smooth muscle myosin will be generated by genetically engineering surface accessible cysteine residues at desired locations in the myosin lever arm and actin binding cleft using a baculovirus/sf9 cell culture expression system. Intramolecular distance measurements in myosin will then be examined by fluorescence resonance energy transfer (FRET), both under bulk solution conditions and at the level of a single molecule, to monitor dynamic structural changes in specific regions of myosin thought to play critical roles in myosin's ability to function as a molecular motor protein. The spectroscopic experiments will be directly correlated in real time with myosin function using stopped-flow measurements to examine the kinetics of the acto-myosin function using stopped-0flow measurements to examine the kinetics of the acto-myosin interaction and an optical laser trap (in collaboration with Dr. David M. Warshaw, Project #2) to examine unitary displacements generated by myosin during active interactions with actin.
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