Troponin is the Ca²⁺ molecular switch that regulates striated muscle contraction. In the heart, troponin Ca²⁺ sensitivity is also modulated by the PKA-dependent phosphorylation of a unique 31-residue N-terminal extension region of the Troponin I subunit (NH₂-TnI). However, the detailed mechanism for the propagation of the phosphorylation signal through Tn, which results in the enhancement of the myocardial relaxation rate, is difficult to examine within whole Tn. Several models exist for how phosphorylation modulates the troponin response in cardiac cells but these are mostly built from peptide-NMR studies and molecular dynamics simulations. Here we used a paramagnetic spin labeling approach to position and track the movement of the NH₂-TnI region within whole Tn. Through paramagnetic relaxation enhancement (PRE)-NMR experiments, we show that the NH₂-TnI region interacts with a broad surface area on the N-domain of the Troponin C subunit. This region includes the Ca²⁺ regulatory Site II and the TnI switch-binding site. Phosphorylation of the NH₂-TnI both weakens and shifts this region to an adjacent site on TnC. Interspin EPR distances between NH₂-TnI and TnC further reveal a phosphorylation induced re-orientation of the TnC N-domain under saturating Ca²⁺ conditions. We propose an allosteric model where phosphorylation triggered cooperative changes in both the interaction of the NH₂-TnI region with TnC, and the re-orientation of the TnC interdomain orientation, together promote the release of the TnI switch-peptide. Enhancement of the myocardial relaxation rate then occurs. Knowledge of this unique role of phosphorylation in whole Tn is important for understanding pathological processes affecting the heart.

肌钙蛋白是调节横纹肌收缩的 Ca ²⁺分子开关。在心脏中，肌钙蛋白 Ca ²⁺敏感性还受到肌钙蛋白 I 亚基 (NH ₂ -TnI)的独特 31 个残基 N 末端延伸区的 PKA 依赖性磷酸化的调节。然而，磷酸化信号通过 Tn 传播的详细机制，导致心肌舒张率的增强，很难在整个 Tn 内检查。存在几种关于磷酸化如何调节心肌细胞肌钙蛋白反应的模型，但这些模型主要是根据肽核磁共振研究和分子动力学模拟建立的。在这里，我们使用顺磁自旋标记方法来定位和跟踪 NH ₂的运动整个 Tn 内的 -TnI 区域。通过顺磁弛豫增强 (PRE)-NMR 实验，我们表明 NH ₂ -TnI 区域与肌钙蛋白 C 亚基 N 域上的广阔表面积相互作用。该区域包括 Ca ²⁺调节位点 II 和 TnI 开关结合位点。NH ₂ -TnI 的磷酸化会削弱该区域并将其转移到 TnC 上的相邻位点。NH ₂ -TnI 和TnC之间的Interspin EPR 距离进一步揭示了在饱和Ca ²⁺条件下磷酸化诱导的TnC N-结构域的重新定向。我们提出了一个变构模型，其中磷酸化触发了 NH ₂相互作用的协同变化-TnI 区域与 TnC，以及 TnC 域间方向的重新定向，共同促进 TnI 开关肽的释放。然后出现心肌舒张率的增强。了解磷酸化在整个 Tn 中的这种独特作用对于理解影响心脏的病理过程很重要。