Schlecht William, Dong Wen-Ji
The Voiland School of Chemical Engineering and Bioengineering and ‡The Department of Integrated Neuroscience and Physiology, Washington State University , Pullman, Washington 99164, United States.
Bioconjug Chem. 2017 Oct 18;28(10):2581-2590. doi: 10.1021/acs.bioconjchem.7b00418. Epub 2017 Sep 18.
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 Ca, cardiac troponin I (cTnI), cardiac troponin T (cTnT), Ca-sensitizers, and a Ca-desensitizing phosphomimic of cTnT (cTnT(T204E) is characterized. Isolated cTnC contained a small open conformation population in the absence of Ca that increased significantly upon the addition of saturating levels of Ca. This suggests that the Ca-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 Ca-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 Ca 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 Ca sensitivity of the heart.
多项研究表明，构象动力学在心肌肌钙蛋白C（cTnC）细丝激活调节中起着重要作用；然而，几乎没有直接证据支持这些说法。在本研究中，开发并实施了一种染料同二聚化方法，该方法可确定cTnC疏水裂缝中开放和封闭构象之间的动态平衡。对这种平衡受钙、心肌肌钙蛋白I（cTnI）、心肌肌钙蛋白T（cTnT）、钙敏化剂以及cTnT的钙脱敏磷酸模拟物（cTnT(T204E)）的调节进行了表征。在没有钙的情况下，分离的cTnC含有少量开放构象群体，在添加饱和水平的钙后，该群体显著增加。这表明钙诱导的细丝激活源于疏水裂缝开放概率的增加。加入cTnI增加了开放cTnC的群体，而加入cTnT则产生相反的效果。与含有野生型心肌肌钙蛋白T [cTnT(wt)]的样品相比，含有钙脱敏cTnT(T204E)的样品开放构象概率略有下降，但不显著，而经钙敏化剂处理的样品通常会增加开放构象概率。这些发现表明，cTnC疏水裂缝开放和封闭构象之间的平衡在调节心脏的钙敏感性方面起着重要作用。
