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

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Dynamic Equilibrium of Cardiac Troponin C’s Hydrophobic Cleft and Its Modulation by Ca2+ Sensitizers and a Ca2+ Sensitivity Blunting Phosphomimic, cTnT(T204E)
Bioconjugate Chemistry ( IF 4.0 ) Pub Date : 2017-09-18 00:00:00 , DOI: 10.1021/acs.bioconjchem.7b00418
William Schlecht ₁ , Wen-Ji Dong ₁

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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.

中文翻译：

钙^离子肌钙蛋白C的疏水性裂口的动态平衡及其对Ca ²⁺增感剂和Ca ²⁺敏感性钝化磷酸cTnT（T204E）的调节

几项研究表明，构象动力学在调节心肌肌钙蛋白C（cTnC）中细丝活化方面很重要。但是，很少有直接证据支持这些说法。在这项研究中，开发并实施了一种染料均二聚方法，该方法可以确定cTnC疏水性裂缝中开放和闭合构象之间的动态平衡。Ca ²⁺，心肌肌钙蛋白I（cTnI），心肌肌钙蛋白T（cTnT），Ca 2 ^+-增敏剂和cTnT（cTnT（T204E）的Ca 2 ^+-脱敏磷酸酯）对该平衡的调节。缺少Ca ²⁺的少量开放构象种群当添加饱和水平的Ca ²⁺时，其显着增加。这表明Ca ²⁺诱导的细丝活化是由于疏水性裂缝开裂的可能性增加而引起的。包含cTnI增加了开放性cTnC的数量，而包含cTnT具有相反的作用。含Ca的样品²⁺ -desensitizing的cTnT（T204E）显示在开放构象的概率轻微但不显着降低相对于心脏肌钙蛋白T的样品，野生型[肌钙蛋白（重量）]，而钙²⁺敏化剂处理过的样品通常会增加开放构象的可能性。这些发现表明，cTnC的疏水性裂缝的开放和闭合构型之间的平衡在调节心脏的Ca ²⁺敏感性中起着重要作用。

更新日期：2017-09-19

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