The long-term goal of this research is to understand the functional and structural consequences of site-specific oxidation in muscle proteins, in order to illuminate the mechanisms by which oxidative stress affects human health and aging. The present proposal focuses on the effect of methionine oxidation in two key muscle proteins - calmodulin (CaM), in its role as regulator of the calcium release channel (ryanodine receptor, RyR) and myosin, in its role as actin-dependent force generator. The rationale for this work comes largely from the previous project period, in which we identified methionine oxidations in CaM and myosin as critical targets of functional decline and protein structural changes in muscle that has been aged or oxidized. In the next period, we focus on fundamental questions about the effects of site-specific methionine oxidation on the structure and function of these two proteins. This project employs site-directed mutagenesis for three purposes: (1) Met mutagenesis will be used to control susceptibility to oxidation, (2) previously identified functional mutations will be introduced to determine their effect on susceptibility to oxidative damage at other sites, (3) Cys mutagenesis will be used to attach spectroscopic probes to selected sites that are designed to detect functionally important structural changes or interactions of CaM or myosin. Thus the functional impacts of specific Met oxidations will be correlated directly with structural impacts. A complementary array of spectroscopic techniques will be used - fluorescence resonance energy transfer (FRET), transient phosphorescence anisotropy (TPA), electron paramagnetic resonance (EPR), and nuclear magnetic resonance (NMR). NMR will allow us to obtain high-resolution structural data on small proteins (CaM) in solution, while the other methods allow us to obtain long-range distance constraints that complement NMR, and to detect structural changes in the large protein complexes in which these proteins function. The high potential impact of this work is made possible by a productive collaboration among three NIH- funded research groups. These groups have demonstrated, through joint publications and preliminary data, the effectiveness of their collaboration in achieving the aims of the previous funding period and establishing feasibility for all aims of the new proposal. This project offers a unique and innovative combination of approaches, all focused on a timely goal - to explain how specific Met oxidations affect muscle protein function, structure, and dynamics. This fundamental information is required for further progress in understanding the structural biology of protein oxidation.