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.

Public Health Relevance

This project's goal is to understand how muscle proteins become damaged by oxidation, the addition of oxygen atoms, which occurs in the processes of aging and degenerative disease. Protein engineering will be done to prevent, mimic, or reverse the oxidation process. 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.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
3R37AG026160-09S2
Application #
8802990
Study Section
Skeletal Muscle Biology and Exercise Physiology Study Section (SMEP)
Program Officer
Williams, John
Project Start
2004-09-15
Project End
2016-03-31
Budget Start
2014-09-15
Budget End
2015-03-31
Support Year
9
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
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Schaaf, Tory M; Peterson, Kurt C; Grant, Benjamin D et al. (2017) Spectral Unmixing Plate Reader: High-Throughput, High-Precision FRET Assays in Living Cells. SLAS Discov 22:250-261
Schaaf, Tory M; Peterson, Kurt C; Grant, Benjamin D et al. (2017) High-Throughput Spectral and Lifetime-Based FRET Screening in Living Cells to Identify Small-Molecule Effectors of SERCA. SLAS Discov 22:262-273
Guhathakurta, Piyali; Prochniewicz, Ewa; Roopnarine, Osha et al. (2017) A Cardiomyopathy Mutation in the Myosin Essential Light Chain Alters Actomyosin Structure. Biophys J 113:91-100
Rebbeck, Robyn T; Nitu, Florentin R; Rohde, David et al. (2016) S100A1 Protein Does Not Compete with Calmodulin for Ryanodine Receptor Binding but Structurally Alters the Ryanodine ReceptorĀ·Calmodulin Complex. J Biol Chem 291:15896-907
Lewis, Andrew K; Dunleavy, Katie M; Senkow, Tiffany L et al. (2016) Oxidation increases the strength of the methionine-aromatic interaction. Nat Chem Biol 12:860-6
Colson, Brett A; Thompson, Andrew R; Espinoza-Fonseca, L Michel et al. (2016) Site-directed spectroscopy of cardiac myosin-binding protein C reveals effects of phosphorylation on protein structural dynamics. Proc Natl Acad Sci U S A 113:3233-8
Avery, Adam W; Crain, Jonathan; Thomas, David D et al. (2016) A human ?-III-spectrin spinocerebellar ataxia type 5 mutation causes high-affinity F-actin binding. Sci Rep 6:21375
Her, Cheng; McCaffrey, Jesse E; Thomas, David D et al. (2016) Calcium-Dependent Structural Dynamics of a Spin-Labeled RyR Peptide Bound to Calmodulin. Biophys J 111:2387-2394
Gomez-Hurtado, Nieves; Boczek, Nicole J; Kryshtal, Dmytro O et al. (2016) Novel CPVT-Associated Calmodulin Mutation in CALM3 (CALM3-A103V) Activates Arrhythmogenic Ca Waves and Sparks. Circ Arrhythm Electrophysiol 9:

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