2015 |
Klein, Jennifer Claire [⬀] Klein, Jennifer Claire [⬀] |
R15Activity Code Description: Supports small-scale research projects at educational institutions that provide baccalaureate or advanced degrees for a significant number of the Nation’s research scientists but that have not been major recipients of NIH support. The goals of the program are to (1) support meritorious research, (2) expose students to research, and (3) strengthen the research environment of the institution. Awards provide limited Direct Costs, plus applicable F&A costs, for periods not to exceed 36 months. This activity code uses multi-year funding authority; however, OER approval is NOT needed prior to an IC using this activity code. |
Structural Impact of Methionine Oxidation in Muscle Proteins @ University of Wisconsin La Crosse
? DESCRIPTION (provided by applicant): 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.
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