Structural and functional effects of oxidative modification of myosin

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The primary goals of the training program are (a) to provide me with extensive training in muscle biochemistry, physiology, and biophysics, toward completion of a PhD thesis in the molecular biophysics of muscle protein oxidation, and (b) to prepare me for the next step along the path to becoming an independent investigator. The long-term goal of the present research program in the Thomas lab is to understand the structural and functional basis of oxidative modification of muscle proteins, in order to determine the molecular basis of decline in muscle strength due to aging and degenerative diseases. Previous work in this lab has shown that (a) aging produces a decline in muscle force, independent of atrophy, accompanied by oxidative modification of myosin, decreasing actin-activated ATPase activity, and (b) peroxide treatment of muscle mimics the functional effects of aging and results primarily in modification of Met residues. I hypothesize that specific Met residues in myosin are responsible for these effects, which result from specific structural perturbations in the catalytic domain of myosin. I will test this hypothesis by performing site-directed Met mutagenesis on myosin in Dictyostelium cell culture, then performing functional and structural tests to determine the correlation between structural dynamics and function. The principal structural approach is site-directed spin labeling (SDSL), followed by electron paramagnetic resonance (EPR). I will pursue three aims. (1) Functional impact of methionine oxidation on myosin catalytic domain. (2) Structural dynamics of myosin catalytic domain. (3) Structural impact of methionine oxidation on the myosin catalytic domain. There is considerable scientific evidence that protein oxidation plays a major role in degenerative disorders associated with aging, but there is very little information about the biochemical and structural effects of oxidation on specific proteins. This project aims to provide a site-specific protein structural explanation for the decline in muscle strength with oxidation, which is hypothesized to play a major role in the loss of muscle strength with age in humans. This advance in fundamental understanding of oxidative mechanisms is important for making progress in treatment of age-related health problems. The approach of using site-directed mutagenesis and spectroscopy to probe the structural basis of oxidative modification in the muscle protein myosin is quite novel. Thus, in addition to providing new structural insight into muscle protein oxidation, this work can serve as a general model for future studies that focus on the specific structural consequences of oxidative stress. This project will build on my past experience in magnetic resonance and aging, while introducing me to a new biological system (muscle) for which the mechanisms of oxidative stress are intensely studied and a new spectroscopic technique (EPR) that is more sensitive than NMR and can be applied under more physiological conditions (e.g., myosin bound to actin).