Beckingham K
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251.
J Biol Chem. 1991 Apr 5;266(10):6027-30.
Mutant versions of the calmodulin of Drosophila melanogaster have been prepared for use in the study of Ca2+ binding and Ca2(+)-induced conformational changes. In each mutant, a conserved glutamic acid residue indicated to play a critical role in Ca2+ binding has been mutated to glutamine in one of the Ca2(+)-binding sites. Thus a series of four proteins, each with an analogous mutation in one of the four binding sites, has been generated. Here the Ca2(+)-induced conformational changes in these proteins have been examined by use of the fluorescent hydrophobic reporter molecule, 9-anthroyl choline. These studies confirm earlier work which indicates that the carboxyl-terminal pair of Ca2(+)-binding sites shows cooperative Ca2+ binding to produce a major conformational change in the protein. However, these studies provide evidence that the sites of the amino-terminal pair are more independent in their Ca2+ binding properties and contribute individually to the conformational changes associated with Ca2+ binding in the amino-terminal half of the protein. This work also indicates that mutation of either of the amino-terminal Ca2(+)-binding sites can influence the conformational change produced by Ca2+ binding to the carboxyl-terminal sites.
已制备出黑腹果蝇钙调蛋白的突变体版本，用于研究钙离子结合及钙离子诱导的构象变化。在每个突变体中，一个在钙离子结合中起关键作用的保守谷氨酸残基在四个钙离子结合位点之一被突变为谷氨酰胺。因此，已产生了一系列四种蛋白质，每种蛋白质在四个结合位点之一具有类似突变。在此，利用荧光疏水报告分子9-蒽甲酰胆碱研究了这些蛋白质中钙离子诱导的构象变化。这些研究证实了早期的工作，即羧基末端的一对钙离子结合位点显示出协同钙离子结合，从而在蛋白质中产生主要的构象变化。然而，这些研究提供了证据，表明氨基末端的一对位点在钙离子结合特性上更独立，并且分别对与蛋白质氨基末端一半中钙离子结合相关的构象变化有贡献。这项工作还表明，氨基末端钙离子结合位点中的任何一个发生突变都可以影响钙离子结合到羧基末端位点所产生的构象变化。
