Dynamic Equilibrium of Cardiac Troponin C's Hydrophobic Cleft and Its Modulation by Ca2+ Sensitizers and a Ca2+ Sensitivity Blunting Phosphomimic, cTnT(T204E).

Several studies have suggested that conformational dynamics are important in the regulation of thin filament activation in cardiac troponin C (cTnC); however, little direct evidence has been offered to support these claims. In this study, a dye homodimerization approach is developed and implemented that allows the determination of the dynamic equilibrium between open and closed conformations in cTnC's hydrophobic cleft. Modulation of this equilibrium by Ca2+, cardiac troponin I (cTnI), cardiac troponin T (cTnT), Ca2+-sensitizers, and a Ca2+-desensitizing phosphomimic of cTnT (cTnT(T204E) is characterized. Isolated cTnC contained a small open conformation population in the absence of Ca2+ that increased significantly upon the addition of saturating levels of Ca2+. This suggests that the Ca2+-induced activation of thin filament arises from an increase in the probability of hydrophobic cleft opening. The inclusion of cTnI increased the population of open cTnC, and the inclusion of cTnT had the opposite effect. Samples containing Ca2+-desensitizing cTnT(T204E) showed a slight but insignificant decrease in open conformation probability compared to samples with cardiac troponin T, wild type [cTnT(wt)], while Ca2+ sensitizer treated samples generally increased open conformation probability. These findings show that an equilibrium between the open and closed conformations of cTnC's hydrophobic cleft play a significant role in tuning the Ca2+ sensitivity of the heart.