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首页> 外文期刊>Biochemistry >Calcium-induced refolding of the calmodulin V136G mutant studied by NMR spectroscopy: evidence for interaction between the two globular domains.
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Calcium-induced refolding of the calmodulin V136G mutant studied by NMR spectroscopy: evidence for interaction between the two globular domains.

机译：钙诱导的钙调蛋白V136G突变体的复性，通过NMR光谱研究：两个球状结构域之间相互作用的证据。

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摘要

The Ca(2+) titration of the (15)N-labeled mutant V136G calmodulin has been monitored using (1)H-(15)N HSQC NMR spectra. Up to a [Ca(2+)]/[CaM] ratio of 2, the Ca(2+) ions bind predominantly to sites I and II on the N-domain in contrast with the behavior of the wild-type calmodulin where the C-terminal domain has the higher affinity for Ca(2+). Surprisingly, the Ca(2+)-binding affinity for the N-domain in the mutant calmodulin is greater than that for the N-domain in the wild-type protein. The mutated C-domain is observed as a mixture of unfolded, partially folded (site III occupied), and native-like folded (sites III and IV occupied) conformations, with relative populations dependent on the [Ca(2+)]/[CaM] ratio. The occupancy of site III independently of site IV in this mutant shows that the cooperativity of Ca(2+) binding in the C-domain is mediated by the integrity of the domain structure. Several NH signals from residues in the Ca(2+)-bound N-domain appear as two signals during the Ca(2+) titration indicating separate species in slow exchange, and it can be deduced that these result from the presence and absence of interdomain interactions in the mutant. It is proposed that an unfolded part of the mutated C-domain interacts with sites on the N-domain that normally bind to target proteins. This would also account for the increase in the Ca(2+) affinity for the N-domain in the mutant compared with the wild-type calmodulin. The results therefore show the wide-ranging effects of a point mutation in a single Ca(2+)-binding site, providing details of the involvement of individual residues in the calcium-induced folding reactions.
机译：已使用（1）H-（15）N HSQC NMR光谱监测（15）N标记的突变型V136G钙调蛋白的Ca（2+）滴定。直到[Ca（2 +）] / [CaM]的比率为2，Ca（2+）离子主要结合到N域的I和II位，与野生型钙调蛋白的行为相反。 C末端域对Ca（2+）具有较高的亲和力。出人意料的是，Ca（2+）结合亲和力的突变钙调蛋白中的N域大于野生型蛋白质中的N域。观察到突变的C结构域是未折叠，部分折叠（位点III占据）和天然折叠（位点III和IV占据）构象的混合物，相对种群取决于[Ca（2 +）] / [ CaM]比。独立于站点IV在此突变体中的站点III的占用情况表明，Ca（2+）在C域中的结合的协同作用是由域结构的完整性介导的。 Ca（2+）绑定的N域中的残留物中的几个NH信号在Ca（2+）滴定过程中显示为两个信号，表明在缓慢的交换中存在不同的物种，可以推断出这些是由于存在或不存在突变体中的域间相互作用。提出了突变的C结构域的未折叠部分与通常结合靶蛋白的N结构域上的位点相互作用。与野生型钙调蛋白相比，这也解释了突变体中N域的Ca（2+）亲和力增加。因此，结果显示了单个Ca（2+）结合位点中的点突变的广泛影响，提供了单个残基参与钙诱导的折叠反应的详细信息。

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