Utilizing Umbrella Sampling and Brownian Dynamics to Study the Functional and Dynamic Characteristics of Cardiac Troponin C

Cardiac troponin C (cTnC) mediates the calcium-induced regulation of muscle contraction through its regulatory, N-terminal domain (cNTnC). Previous work utilizing umbrella sampling and molecular dynamics simulations on this domain of cTnC has shown mutations associated with hypertrophic (HCM) and dilated cardiomyopathies (DCM) influence the free energy of opening which suggested a mechanism for altering calcium sensitivity. To elaborate on the study of the dynamic features of cTnC, other computational methods are used here to investigate the energetics of calcium binding and troponin-I switch peptide binding. We used BrownDye and umbrella sampling to calculate calcium on-rates and free energies of binding, respectively. Results of these simulations have shown our ability to recapitulate experimental data. Small molecules have also been previously shown to bind to cTnC's hydrophobic patch region and modulate calcium sensitivity as a potential therapeutic for cardiomyopathies associated with mutations in this region of cNTnC. The umbrella sampling protocol developed for studying HCM/DCM associated mutations, as well as molecular dynamics simulations, are used here to investigate a potential mechanism of these small molecules for altering calcium sensitivity. We were able to show that the addition of small molecules in the hydrophobic patch caused a lowering of the free energy of opening and a stabilization of a semi-open configuration. These methodologies for studying cTnC will provide a holistic view of the dynamics and functions of cTnC facilitating a molecular understanding of cardiac muscle contraction.