Dobrowolski Z, Xu G Q, Chen W, Hitchcock-DeGregori S E
Department of Neuroscience and Cell Biology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway 08854.
Biochemistry. 1991 Jul 23;30(29):7089-96. doi: 10.1021/bi00243a008.
Five deletion mutants of the D/E linker region of the troponin C central helix were tested for conformational and functional differences from wild-type troponin C. The mutants were in the region 87KEDAKGKSEEE97: dEDA, dKG, dKGK, dKEDAKGK, and dSEEE, designed to change the length of the central helix and the orientation of the Ca(2+)-binding domains relative to each other [Dobrowolski, Z., Xu, G.-Q., & Hitchcock-DeGregori, S.E. (1991) J. Biol. Chem. 266, 5703-5710]. Previous work showed that all mutants except dSEEE are partially defective in one part of the Ca2+ switch or the other. All mutants undergo Ca(2+)-dependent conformational changes as detected by changes in electrophoretic mobility, alpha-helix content, and hydrophobic exposure. Deletions of the central helix do not extensively alter the thermal stability of troponin C, as determined by temperature-dependent loss of alpha-helix. There are differences among the mutants that do not correlate with function. All troponin C mutants show Ca(2+)-dependent interaction with troponin I and T in polyacrylamide gels. Troponin I-troponin C interaction was also analyzed by Ca(2+)-dependent increase in the monomer/excimer ratio of tropinin I and relief of inhibition of the actomyosin S1 ATPase. While all mutants retain basic function, dKGK, dKEDAKGK, and dEDA have altered interaction with troponin I in the absence of Ca2+. dSEEE differs in conformation from wild type, but it is normal in functional assays. This conserved region of the D/E linker is not required for interaction with troponin I in the presence or absence of urea.
对肌钙蛋白C中央螺旋D/E连接区的五个缺失突变体进行了测试，以研究其与野生型肌钙蛋白C在构象和功能上的差异。这些突变体位于87KEDAKGKSEEE97区域：dEDA、dKG、dKGK、dKEDAKGK和dSEEE，旨在改变中央螺旋的长度以及Ca(2+)结合结构域相对于彼此的方向[多布罗沃尔斯基，Z.，徐，G.-Q.，&希区柯克-德格雷戈里，S.E.（1991年）《生物化学杂志》266，5703 - 5710]。先前的研究表明，除dSEEE外，所有突变体在Ca2+开关的某一部分存在部分缺陷。通过电泳迁移率、α - 螺旋含量和疏水暴露的变化检测到，所有突变体都经历了Ca(2+)依赖性的构象变化。如通过α - 螺旋随温度的丧失所确定的，中央螺旋的缺失并未广泛改变肌钙蛋白C的热稳定性。突变体之间存在与功能不相关的差异。在聚丙烯酰胺凝胶中，所有肌钙蛋白C突变体都显示出与肌钙蛋白I和T的Ca(2+)依赖性相互作用。还通过肌钙蛋白I的单体/准分子比率的Ca(2+)依赖性增加以及肌动球蛋白S1 ATP酶抑制的解除来分析肌钙蛋白I与肌钙蛋白C的相互作用。虽然所有突变体都保留了基本功能，但dKGK、dKEDAKGK和dEDA在无Ca2+时与肌钙蛋白I的相互作用发生了改变。dSEEE在构象上与野生型不同，但在功能测定中是正常的。在有或没有尿素的情况下，D/E连接区的这个保守区域对于与肌钙蛋白I的相互作用不是必需的。
