Dong W J, Xing J, Chandra M, Solaro J, Cheung H C
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, 35294-2041, USA.
Proteins. 2000 Dec 1;41(4):438-47.
The global conformation of cardiac muscle troponin I (cTnI) was investigated with single-cysteine mutants by using a combination of sulfhydryl reactivity and fluorescence resonance energy transfer (FRET) to determine cysteine accessibility and intersite distances. The reactivity was determined with a fluorescent reagent for its reaction with cysteine residues singly located at positions 5, 40, 81, 98, 115, 133, 150, 167, and 192. FRET measurements were made by using the endogenous single Trp-192 as the energy donor and an acceptor probe covalently attached to the cysteines as energy acceptor. The results suggest an open and extended conformation of cTnI with a large curvature in which the cysteines are highly exposed to the solvent. These conformational features are largely retained in the segment between residues 40 and 192 upon phosphorylation at Ser-23 and Ser-24. The sulfhydryl groups of the Cys-133 and Cys-150 of the cTnI incorporated into the binary cTnC-cTnI and fully reconstituted troponin complexes experience large reduced exposure resulting from the binding of Ca(2+) to the regulatory site of cTnC, suggesting that key regions of cTnI involved in activation become highly shielded upon activation. In the cTnC-cTnI complex, every intramolecular distance in the cTnI is lengthened and the overall conformation of the bound cTnI remains elongated with reduced exposure for the cysteines. The global conformation of the troponin C-troponin I complex from cardiac muscle has an elongated shape with constrained flexibility. The highly flexible nature of the N-terminal extension of cTnI is preserved in the complex, suggesting that this segment of cTnI is either not bound or only loosely bound to the C-domain of cTnC.
通过单半胱氨酸突变体，结合巯基反应性和荧光共振能量转移（FRET）来测定半胱氨酸可及性和位点间距离，从而研究心肌肌钙蛋白I（cTnI）的整体构象。使用一种荧光试剂测定其与分别位于5、40、81、98、115、133、150、167和192位的单个半胱氨酸残基的反应性。FRET测量是以内源性的单个色氨酸-192作为能量供体，以及一个与半胱氨酸共价连接的受体探针作为能量受体进行的。结果表明cTnI具有开放且伸展的构象，曲率较大，其中半胱氨酸高度暴露于溶剂中。在丝氨酸-23和丝氨酸-24磷酸化后，这些构象特征在40至192位残基之间的片段中基本保留。掺入二元cTnC-cTnI和完全重组肌钙蛋白复合物中的cTnI的半胱氨酸-133和半胱氨酸-150的巯基由于Ca(2+)与cTnC调节位点的结合而暴露大幅减少，这表明cTnI中参与激活的关键区域在激活后变得高度屏蔽。在cTnC-cTnI复合物中，cTnI中的每个分子内距离都延长，结合的cTnI的整体构象仍然拉长，半胱氨酸的暴露减少。心肌肌钙蛋白C-肌钙蛋白I复合物的整体构象呈细长形，柔韧性受限。cTnI N端延伸的高度柔韧性在复合物中得以保留，这表明cTnI的这一片段要么未与cTnC的C结构域结合，要么只是松散结合。
