Calcium-Induced Refolding of the Calmodulin V136G Mutant Studied by NMR Spectroscopy: Evidence for Interaction between the Two Globular Domains

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• 作者：Sandrine Fefeu ; Rodolfo R. Biekofsky ; John E. McCormick ; Stephen R. Martin ; Peter M. Bayley ; and James Feeney
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：December 26, 2000
• 年：2000
• 卷：39
• 期：51
• 页码：15920 - 15931
• 全文大小：136K
• 年卷期：v.39,no.51(December 26, 2000)
• ISSN：1520-4995

文摘

The Ca²⁺ titration of the ¹⁵N-labeled mutant V136G calmodulin has been monitored using¹H-¹⁵N HSQC NMR spectra. Up to a [Ca²⁺]/[CaM] ratio of 2, the Ca²⁺ ions bind predominantly to sitesI and II on the N-domain in contrast with the behavior of the wild-type calmodulin where the C-terminaldomain has the higher affinity for Ca²⁺. Surprisingly, the Ca²⁺-binding affinity for the N-domain in themutant calmodulin is greater than that for the N-domain in the wild-type protein. The mutated C-domainis observed as a mixture of unfolded, partially folded (site III occupied), and native-like folded (sites IIIand IV occupied) conformations, with relative populations dependent on the [Ca²⁺]/[CaM] ratio. Theoccupancy of site III independently of site IV in this mutant shows that the cooperativity of Ca²⁺ bindingin the C-domain is mediated by the integrity of the domain structure. Several NH signals from residuesin the Ca²⁺-bound N-domain appear as two signals during the Ca²⁺ titration indicating separate speciesin slow exchange, and it can be deduced that these result from the presence and absence of interdomaininteractions in the mutant. It is proposed that an unfolded part of the mutated C-domain interacts withsites on the N-domain that normally bind to target proteins. This would also account for the increase inthe Ca²⁺ affinity for the N-domain in the mutant compared with the wild-type calmodulin. The resultstherefore show the wide-ranging effects of a point mutation in a single Ca²⁺-binding site, providing detailsof the involvement of individual residues in the calcium-induced folding reactions.