Abstract

Cardiac Troponin C (TnC) is a Ca2+-binding protein and plays an important role in regulation of muscle contraction. Mutations in cTnC are implicated in phenotypic characteristics known as hypertrophic and dilated cardiomyopathy (HCM and DCM, respectively). However, the structural mechanisms underlying cardiac dysfunction are unknown. The main goal of this work is to investigate changes in stability and dynamics of seven cTnC variants (A8V, D145E, C84Y and A31S related to HCM; and Y5H, M103I and I148V related to DCM) using an ensemble of thermodynamic and structural approaches. Ca2+-titrations monitored by bis-ANS fluorescence revealed that D145E, A31S and all mutations related with DCM decreased the Ca2+-induced hydrophobic exposure, while C84Y substantially enhance it by the N-domain exposure compared to WT. Thermostability monitored by circular dichroism revealed similar melting temperatures between apo and holo states for D145E (apo: 66.4 ± 1.4°C, holo: 65.4 ± 1.6°C) but different values for WT (apo: 65 ± 1.9°C and holo: >90°C) and C84Y (apo: 43.8 ± 1.5°C, holo: 66.6 ± 0.8°C). Shape restorations from small angle X-ray scattering were used to evaluate conformational changes induced by the mutations. In the apo state, an increase in the radius of gyration values upon increasing concentrations of urea was observed for the Y5H, A31S, M103I and I148V mutants compared to WT. Furthermore, the D145E displayed the most affected shape compared to WT and perturbed residues were located at the C-domain as confirmed by chemical shift perturbation analysis. In addition, the D145E secondary structure was not significantly altered by dihedral angles prediction from the NMR assignment data. These observations open up new avenues for the comprehension of the complex behavior of HCM and DCM mutations in cTnC that has heretofore been not evaluated at structural level.

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