Ca2+ binding and conformational change in two series of point mutations to the individual Ca(2+)-binding sites of calmodulin.
Ca2+ binding and conformational change in two series of point mutations to the individual Ca(2+)-binding sites of calmodulin.
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DOI：
10.1016/s0021-9258(18)42764-0
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发表时间：
1992-03
期刊：
影响因子：
--
通讯作者：
J. Maune;C. Klee;K. Beckingham
中科院分区：
文献类型：
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作者：
J. Maune;C. Klee;K. Beckingham
对果蝇钙调素个体钙结合位点的两系列定点突变进行了研究。在每个突变体中，所有Ca(2+)结合环中第12位的保守谷氨酸残基在一个位置发生了突变。在一个系列中，残基被改变为谷氨酰胺；在第二系列中，该残基被改变为赖氨酸。给出了这些突变体和野生型蛋白在拟生理条件下的钙结合特性。此外，还研究了Ca(2+)对部分突变体的单一酪氨酸残基(Tyr-138)环境的影响。与野生型蛋白结合的最佳模型是两对位点，具有较高的亲和力对，显示出很强的协作性。在这8种突变蛋白中，除1种外，仅能检测到3种钙结合事件。在三个氨基端突变体中，三个残留点是(I)一对相对高亲和力的位点和(Ii)一个弱化的低亲和力位点。对于所有四个羧基末端突变，残留点是三个相对低亲和力的位点。总体而言，位点2和4的突变比位点1和3的突变更有害。Ca(2+)诱导的Tyr-138附近的构象变化相对不受位点1的突变的干扰。然而，羧基末端突变体中Tyr-138的变化表明，当高亲和力位点的协同结合被破坏时，羧基末端的构象变化发生在两个阶段。似乎钙离子与任何一个羧基末端结合都能引起第一阶段的反应，但当第四位点为突变位点时，第二阶段几乎被取消。因此，第3位和第4位突变体的最终构象明显不同。
Two series of site-directed mutations to the individual Ca(2+)-binding sites of Drosophila melanogaster calmodulin have been generated and studied. In each mutant, a conserved glutamic acid residue at position 12 in all of the Ca(2+)-binding loops has been mutated in one site. In one series the residue is changed to glutamine; in the second series the change is to lysine. The Ca(2+)-binding properties of these mutants and the wild-type protein under pseudo-physiological conditions are presented. In addition, Ca(2+)-induced changes to the environment of the single tyrosine residue (Tyr-138) have been studied for some of the mutants. Ca2+ binding to the wild-type protein is best modeled as two pairs of sites with a higher affinity pair that shows strong cooperativity. For all but one of these eight mutant proteins, only three Ca(2+)-binding events can be detected. In three of the amino-terminal mutants, the three residual sites are (i) a pair of relatively high affinity sites and (ii) a weakened low affinity site. For all four carboxyl-terminal mutations, the residual sites are three relatively low affinity sites. In general, mutations to sites 2 and 4 prove more deleterious than mutations to sites 1 and 3. The Ca(2+)-induced conformational changes in the vicinity of Tyr-138 are relatively undisturbed by mutations of site 1. However, the changes to Tyr-138 in the carboxyl-terminal site mutants indicate that upon disruption of the cooperative binding at the high affinity sites, conformational change in the carboxyl terminus occurs in two phases. It appears that binding of Ca2+ to either carboxyl-terminal site can elicit the first phase of the response but the second phase is almost abolished when site 4 is the mutated site. The final conformations of site 3 and 4 mutants are thus significantly different.