Genetic screening has detected abundant mutations in sarcomeric proteins elucidating basic causes for disease and identifying targets for individualized medicine when a functional deficit on the protein level can be identified. The project focuses on the molecular motor myosin and its regulation using various approaches for the expression, dynamical characterization, and structural visualization of the protein in its native and mutated forms. The goal is to decipher the role individual mutations play in modifying native myosin function. Myosin performs ATP free energy transduction into mechanical work by coordinating ATP hydrolysis at the active site, actin affinity modulation at the actin binding site, and the lever-arm power stroke, via allosteric transduction pathways operating in a time ordered sequence. Energy transduction is the definitive systemic feature of myosin and a working model for native transduction allocates specific functions to structural domains within the motor beginning with ATP hydrolysis in the active site and ending in a power stroke rotating a lever- arm domain in the motor through ~70 degrees in the crowded environment of the muscle tissue. The cardiac myosin heavy chain (MHC) and both of its light chains (MLCs) harbor familial hypertrophic cardiomyopathy (FHC)-linked mutations. MHC mutants are hypothesized to disrupt specific transduction pathways. Evolutionarily conserved allosteric connectivity prediction identifies residues in MHC forming the transduction pathway. Transduction pathway residues that are also FHC-linked mutation sites identify the MHC candidate mutants affecting transduction. Several MLC mutants are hypothesized to impact lever-arm structural stability influencing lever-arm dynamics and effectiveness. Myosin modified by a disease-linked MHC or MLC candidate mutation is subjected to in vitro and in situ experiments to determine how the mutations impact, the functional domains in MHC operating in a working model for native transduction, or the lever-arm stability provided by the MLC. A single molecule experiment detecting lever-arm rotary movement is especially pertinent because it is applicable to myosin in the native crowded environment of the muscle fiber. Myosin regulatory light chain (RLC) may have special significance because it is partially phosphorylated at Ser15 in normal cardiac tissue. Phosphorylation apparently affects myosin calcium regulation while in the muscle tissue and myosin duty ratio in vitro within single myosin motors. In the latter case, RLC conformation modulation by phosphorylation must impact myosin function related to strong actin binding. RLC crystallization and structure determination will investigate the structural basis of RLC regulation of myosin as well as the impact of FHC-linked mutations on RLC structure.

Public Health Relevance

Familial hypertrophic cardiomyopathy (FHC) is a disease characterized by an enlarged heart. It affects 1 in 500 persons and is a cause of sudden cardiac death in the young. Genetic mutations affecting protein structure and function in the heart are linked to FHC. The project goal is to associate the mutation with the specific protein function affected to identify basic causes for disease and targets for individualized medicine. The proposed research applies promising new computational and experimental approaches for assessing how disease implicated mutations change the motor powering contraction.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL095572-04
Application #
8457105
Study Section
Cardiac Contractility, Hypertrophy, and Failure Study Section (CCHF)
Program Officer
Adhikari, Bishow B
Project Start
2010-07-15
Project End
2014-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
4
Fiscal Year
2013
Total Cost
$371,573
Indirect Cost
$135,953
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
006471700
City
Rochester
State
MN
Country
United States
Zip Code
55905
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