): The focus of this proposal is to study the conformational changes within the actomyosin complex using new forms of fluorescence energy transfer (FRET) developed in my laboratory, combined with genetically engineered """"""""cysteine-light"""""""" myosins. Specifically the FRET techniques are lanthanide-based resonance energy transfer (LRET), single-molecule FRET (smFRET), and fluorescence fluctuation spectroscopy FRET (FFS-FRET). Together, these techniques provide unique abilities to measure structures in the 20-100 A range with Angstrom precision, dynamics over a wide range, i.e., more than 1 usec, and with single-molecule sensitivity. While the focus is on actomyosin, these techniques are applicable to many protein systems, and indeed we have used them to recently detect conformational changes in ion channels. The specific myosins will be smooth muscle myosin II, because of its clinical importance - it forms the lining of blood vessels, uterus, gut - and later, myosin VI, recently shown to be a """"""""backwards-moving"""""""" myosin. Cys-light proteins enable us to probe conformational changes throughout the large actomyosin protein complex, and to mutate the protein to emphasize certain states of the catalytic cycle. Myosin is a dimeric molecular motor in which conformational changes following ATP hydrolysis leads to translation of actin filaments, leading to muscle contraction and a variety of subcellular motion in eucaryotes. The major goal of this project to understand the structure and function of the myosin dimer, and in doing so understand smooth muscle regulation at the molecular level. (Regulation requires a dimer and is controlled via phosphorylation.) This will give insight into fundamental questions such as: Why is myosin a dimer? How tightly are the two heads coupled together? How does the dimer interact with actin? How does phosphorylation at one end of the myosin head turn on the ATPase at the other end, over 7 nm away? To do this we will measure distances (LRET, FRET), distribution of distances (LRET, smFRET), and dynamics (LRET, FRET, FFS-FRET) within each head of the dimer, between the heads, and between the heads and the S2-rod which holds the two heads together. Measurements on single headed myosin (SI), particularly as a function of actin, will also be performed as controls and to help test the """"""""lever-arm"""""""" model of muscle mechanics. Specific questions underlying the lever arm model will be answered: 1) Does actin open a cleft in myosin head to activate its ATPase? Does the ADP-swing of myosin, possibly associated with the physiologically important """"""""latch"""""""" state, depend on actin? Does the rod uncoil upon two-headed binding of myosin to actin? Finally, FRET measurements on Myosin VI will be performed to determine the structures and kinetics associated with backwards motion, including the effect of actin and nucleotides. This new-and apparently unique-myosin remains largely uncharacterized. These measurements follow those we and others have made on myosin II.
