My research is focused on skeletal muscle myosin and its interaction with actin filaments. As an undergraduate, I do most work in collaboration with Dr. Joseph M. Muretta. We use an in vitro model of muscle with myosin harvested from the amoeba Dictyostelium discoideum and actin harvested from rabbit skeletal muscle. To investigate structural changes in myosin, we exploit structural models to design labeling sites which are then created by site-directed mutagenesis of the myosin gene. Once purified, this myosin is labeled with various fluorescent probes. We then use FRET to measure distances within myosin during its enzymatic cycle. Current work uses a pair of probes on a structure called the relay helix. This helix bends during the myosin powerstroke, causing the distance between probes to decrease.
We combine this structural information with kinetics to generate kinetic models describing muscle contraction at the molecular level. To further develop the structural kinetics of myosin, we use phosphate-binding protein (PBP), a protein secreted by E. coli. When labeled with a fluorescent probe, PBP becomes an effective sensor for phosphate release at rates seen during the myosin enzymatic cycle (~20/s). We are also able to measure the rate of myosin binding to actin by labeling actin with pyrene. In this way we measure three events during the myosin powerstroke: relay helix bending, phosphate release and relay helix bending.
In support of this work on myosin, we also develop technology to acquire and process fluorescence data from the "Fargoland" transient time-resolved spectrometer. This (TR)2 instrument uses a pulsed laser to measure fluorescence decays on the ns time scale. By integrating these decays, a ms scale measurement is obtained allowing us to compare our novel (TR)2 instrument with conventional instruments. In addition, we are developing methods for use with the ns scale microplate-based spectrometer "NovaFluor" (Fluorescence Innovations, Inc.). The microplate format is exciting because it will permit us to map any experiment onto a 2-dimensional concentration gradient, potentially accelerating discovery.