Our goal is to explain in molecular detail how age-related muscle degeneration is related to the oxidation of muscle proteins by biological oxidants, from atomic-level changes in protein structure, to changes in protein regulatory interactions, and finally, to biological aging on the organismal level. The general hypothesis is that site-specific oxidative modifications of the amino acids methionine (Met) and cysteine (Cys) trigger changes in muscle protein function by altering protein structural dynamics. This advance in fundamental understanding of oxidative mechanisms is important for making progress in treating a broad range of human degenerative diseases, not just muscle.
Aim 1 involves the proteins directly involved in muscle contraction, myosin and actin, and focuses on the key element of force generation: actomyosin interaction.
Aim 2 involves key proteins involved in muscle regulation, calmodulin (CaM) and the ryanodine receptor complex (RyR), and focuses on the CaM/RyR structural interaction that modulates calcium release. The rationale for this work comes, in part, from the investigator's previous work, in which Met oxidations in myosin and CaM were identified as critical targets of functional decline and protein structural changes in muscle that has been aged or oxidized. We focus on fundamental questions about the effects of site-specific oxidation on the structure and function of CaM and myosin. This project employs site-directed mutagenesis for two purposes: (1) Met and Cys mutagenesis will be used to control susceptibility to oxidative modifications, and (2) Cys mutagenesis will be used to attach spectroscopic probes to selected sites that are designed to detect functionally critical structural changes or interactions of CaM or myosin. Thus the functional impacts of specific oxidations, including aging biomarkers, will be correlated directly with structural impacts. A complementary array of spectroscopic techniques will be used - fluorescence resonance energy transfer (FRET), electron paramagnetic resonance (EPR), and nuclear magnetic resonance (NMR). NMR will allow us to obtain high-resolution structure and dynamics 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 productive collaboration between investigators at small, primarily undergraduate institutions, and larger research-driven institutions. These groups have demonstrated, through joint publications and preliminary data, the effectiveness of their collaboration. This project offers a unique and innovative combination of approaches, all focused on a timely goal - to explain how specific oxidative modifications affect muscle protein function, structure, and dynamics. This fundamental information is required for further progress in understanding the structural biology of protein oxidation, and therefore the molecular basis of aging and age-related muscle dysfunction.
This project's goal is to understand how muscle proteins are structurally poised to sense, respond and eventually become damaged by oxidation, which occurs in the processes of aging and degenerative disease. This fundamental information will inform future efforts to prevent or reverse the effects of aging and degenerative diseases such as heart failure and muscle weakness.
| Walgenbach, Daniel G; Gregory, Andrew J; Klein, Jennifer C (2018) Unique methionine-aromatic interactions govern the calmodulin redox sensor. Biochem Biophys Res Commun 505:236-241 |
| McCarthy, Megan R; Thompson, Andrew R; Nitu, Florentin et al. (2015) Impact of methionine oxidation on calmodulin structural dynamics. Biochem Biophys Res Commun 456:567-72 |
