Effects of PKA phosphorylation of Cardiac troponin I and strong crossbridge on conformational transitions of the N-domain of Cardiac troponin C in regulated thin filaments
Effects of PKA phosphorylation of Cardiac troponin I and strong crossbridge on conformational transitions of the N-domain of Cardiac troponin C in regulated thin filaments
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DOI：
10.1021/bi700574n
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发表时间：
2007-08-28
期刊：
影响因子：
2.9
通讯作者：
Cheung, Herbert C.
中科院分区：
文献类型：
--
作者：
Dong, Wen-Ji;Jayasundar, Jayant James;Cheung, Herbert C.
心肌功能的调节是通过Ca 2+与肌钙蛋白C（cTnC）结合而启动的，其诱导cTnC和其他细丝蛋白的一系列结构变化。这些结构的变化进一步调制的横桥形成和微调的磷酸化cTnI。本研究的目的是使用一种新的基于Forster共振能量转移的结构标记物来区分Ca 2+结合、跨桥相互作用和cTnI的蛋白激酶A磷酸化对cTnC N-结构域构象变化的结构和动力学影响。通过将AEDANS连接到作为FRET供体的双半胱氨酸突变体cTnC（13 C/51 C）的一个半胱氨酸并将DDPM连接到作为受体的另一个半胱氨酸来产生基于FRET的结构标记。通过加入cTnI、cTnT、原肌球蛋白和肌动蛋白，将双标记cTnC突变体重组为细丝。Cys 13和Cys 51之间的距离的变化诱导的Ca 2+结合/解离的FRET感应Ca 2+滴定和停流研究，和时间分辨荧光测量。结果表明，钙离子和肌球蛋白头与肌动蛋白的强结合的存在下，需要实现一个完全开放的结构的cTnC N-域在调节细丝。平衡和停流研究表明，强结合的肌球蛋白头显着增加的Ca 2+的敏感性，改变cTnC N-域的结构转变的动力学。cTnI的PKA磷酸化影响cTnC N-结构域结构转变的Ca 2+敏感性和动力学，但对cTnC开放没有显示出全局结构效应。这些结果提供了一个深入了解的调制机制，强交联和肌钙蛋白I磷酸化在心脏细丝激活/舒张过程。
Regulation of cardiac muscle function is initiated by binding of Ca2+ to troponin C (cTnC) which induces a series of structural changes in cTnC and other thin filament proteins. These structural changes are further modulated by crossbridge formation and fine-tuned by phosphorylation of cTnI. The objective of the present study is to use a new Forster resonance energy transfer-based structural marker to distinguish structural and kinetic effects of Ca2+ binding, crossbridge interaction, and protein kinase A phosphorylation of cTnI on the conformational changes of the cTnC N-domain. The FRET-based structural marker was generated by attaching AEDANS to one cysteine of a double-cysteine mutant cTnC(13C/51C) as a FRET donor and attaching DDPM to the other cysteine as the acceptor. The doubly labeled cTnC mutant was reconstituted into the thin filament by adding cTnI, cTnT, tropomyosin, and actin. Changes in the distance between Cys13 and Cys51 induced by Ca2+ binding/dissociation were determined by FRET-sensed Ca2+ titration and stopped-flow studies, and time-resolved fluorescence measurements. The results showed that the presence of both Ca2+ and strong binding of myosin head to actin was required to achieve a fully open structure of the cTnC N-domain in regulated thin filaments. Equilibrium and stopped-flow studies suggested that strongly bound myosin head significantly increased the Ca2+ sensitivity and changed the kinetics of the structural transition of the cTnC N-domain. PKA phosphorylation of cTnI impacted the Ca2+ sensitivity and kinetics of the structural transition of the cTnC N-domain but showed no global structural effect on cTnC opening. These results provide an insight into the modulation mechanism of strong crossbridge and cTnI phosphorylation in cardiac thin filament activation/relaxation processes.