Dissection of the Pathway of Molecular Recognition by Calmodulin

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• 作者：James K. Kranz ; Peter F. Flynn ; Ernesto J. Fuentes ; and A. Joshua Wand
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：February 26, 2002
• 年：2002
• 卷：41
• 期：8
• 页码：2599 - 2608
• 全文大小：203K
• 年卷期：v.41,no.8(February 26, 2002)
• ISSN：1520-4995

文摘

Amide hydrogen exchange has been used to examine the structural dynamics and energeticsof the interaction of a peptide corresponding to the calmodulin-binding domain of smooth muscle myosinlight chain kinase (smMLCKp) with calcium-saturated calmodulin. Heteronuclear NMR ¹⁵N-¹H correlationspectroscopy was used to quantify amide proton exchange rates of the uniformly ¹⁵N-labeled domainbound to calmodulin. A key feature of a proposed model for molecular recognition by calmodulin [Ehrhardtet al. (1995) Biochemistry 34, 2731-2738] is tested by examination of the dependence of amide hydrogenexchange on applied hydrostatic pressure. Hydrogen exchange rates and corresponding protection factors(1/K_op) for individual amide protons of the bound smMLCKp domain span 5 orders of magnitude atambient pressure. Individual protection factors decrease significantly in a linear fashion with increasinghydrostatic pressure. A common pressure dependence is revealed by a constant large negative volumechange across the residues comprising the core of the bound helical domain. The pattern of protectionfactors and their response to hydrostatic pressure is consistent with a structural reorganization that resultsin the concerted disruption of ion pairs between calmodulin and the bound domain. These observationsreinforce a model for the molecular recognition pathway where formation of the initial encounter complexis followed by helix-coil transitions in the bound state and subsequent concerted formation of the extensiveion pair network defining the intermolecular contact surface between CaM and the target domain in thefinal, compact complex structure.