Osha Roopnarine, Principal Investigator

Correlation of EPR Spectroscopic Dynamics and Computer Simulated Molecular Dynamics of Mutations That Cause Heart Disease

Several different point mutations in the myosin heavy chain and light chains (LC) cause the human heart disease known as familial hypertrophic cardiomyopathy (FHC). The goal of this research was to understand the biophysical and biochemical basis of the mutant phenotypes by performing site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy on FHCmutant RLC in muscle fibers. The research is intended to determine the effect of the FHC mutations on the protein structure by simulating the mutations in crystal structures of myosin to ascertain whether or not the tertiary structure of the protein is affected by the mutation. Using the molecular modeling program INSIGHTII to construct energy-minimized models for the LC domain containing these rat VRLC mutations, this researcher planned to engineer cysteine residues on the crystal structures of the protein to determine if it is an appropriate site for minimal protein structural perturbation. This would allow development of an experimental design of spectroscopic studies. The simulated Cys mutants would then be used as the background for the FHC mutations. Following this work, the molecular dynamics program was expected to simulate the dynamics of a spin label attached to the Cys, in order to determine if the site for labeling would detect specific motional changes within the protein.

Preliminary work with the FHC-RLC mutations allowed the visualization of probable structures and the performance of molecular dynamics simulations to be compared directly with EPR spectroscopic data. The results of this were expected to suggest that the FHC mutation perturbs the flexibility of the region that connects the two principal lobes of the RLC.