Troponin C (TnC) facilitates muscle contraction through calcium-binding within its N-terminal region (NTnC). This calcium-binding event leads to an increase in the dynamics of helices lining a hydrophobic patch on TnC. We applied umbrella sampling to probe the TnC hydrophobic patch opening in a targeted and quantitative fashion. We investigated the differences in the free energy of opening between cardiac (cTnC) and fast skeletal TnC (sTnC) and found that, in agreement with previous reports, holo (calcium-bound) sTnC had a lower free energy of opening compared with holo cTnC. Additionally, differences in the free energy of opening of hypertrophic (HCM) and dilated cardiomyopathy (DCM) cTnC systems were investigated. MD simulations and umbrella sampling revealed a lower free energy of opening for the HCM mutations A8V and A31S, as well as the calcium-sensitizing mutation L48Q. The DCM mutations, Y5H, Q50R, and E59D/D75Y, all exhibited a higher free energy of opening. An umbrella sampling simulation of cTnI-bound holo cTnC exhibited the lowest free energy in the open configuration, in agreement with experimental data. Additionally, small molecules have also been 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, were used 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.

Molecular dynamics and umbrella sampling simulations elucidate differences in troponin C isoform and mutant hydrophobic patch exposure

2019 Spring National Meeting

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