Use of site-directed mutations in the individual Ca2(+)-binding sites of calmodulin to examine Ca2(+)-induced conformational changes.
Use of site-directed mutations in the individual Ca2(+)-binding sites of calmodulin to examine Ca2(+)-induced conformational changes.
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DOI：
10.1016/s0021-9258(18)38077-3
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发表时间：
1991-04
期刊：
影响因子：
--
通讯作者：
K. Beckingham
中科院分区：
文献类型：
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作者：
K. Beckingham
已制备了果蝇钙调素的突变体，用于研究Ca ~（2+）结合和Ca ~（2+）诱导的构象变化。在每个突变体中，在Ca 2+结合中起关键作用的保守谷氨酸残基在其中一个Ca 2（+）结合位点突变为谷氨酰胺。因此，已经产生了一系列的四种蛋白质，每种蛋白质在四个结合位点之一具有类似的突变。在这里，Ca 2（+）诱导的构象变化，这些蛋白质已被检查通过使用荧光疏水报告分子，9-蒽酰胆碱。这些研究证实了早期的工作，表明羧基末端的Ca 2（+）结合位点对显示出协同的Ca 2+结合，从而在蛋白质中产生主要的构象变化。然而，这些研究提供的证据表明，氨基末端对的网站是更独立的Ca 2+结合特性，并单独贡献的构象变化与Ca 2+结合在氨基末端的一半的蛋白质。这一工作还表明，氨基端的Ca ~（2+）结合位点的任何一个突变都可以影响Ca ~（2+）结合羧基端位点所产生的构象变化。
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.