The Transition State of Coupled Folding and Binding for a Flexible 尾-Finger

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• 作者：O. Andreas Karlsson^&dagger ; ; Celestine N. Chi^&dagger ; ; Å ; ke Engstr&ouml ; m ; Per Jemth ; ^{Per.Jemth@imbim.uu.se}
• 关键词：nNOS ; neuronal nitric oxide synthase
• 刊名：Journal of Molecular Biology
• 出版年：2012
• 期刊代码：102_00222836
• 类别：imm
• 出版时间：30 March, 2012
• 卷：417
• 期：3
• 页码：253-261
• 文件大小：498 K

摘要

Flexible and fully disordered protein regions that fold upon binding mediate numerous protein-protein interactions. However, little is known about their mechanism of interaction. One such coupled folding and binding occurs when a flexible region of neuronal nitric oxide synthase adopts a 尾-finger structure upon binding to its protein ligand, a PDZ [PSD-95 (postsynaptic density protein-95)/Discs large/ZO-1] domain from PSD-95. We have analyzed this binding reaction by protein engineering combined with kinetic experiments. Mutational destabilization of the 尾-finger changed mainly the dissociation rate constant of the proteins and, to a lesser extent, the association rate constant. Thus, mutation affected late events in the coupled folding and binding reaction. Our results therefore suggest that the native binding interactions of the 尾-finger are not present in the rate-limiting transition state for binding but form on the downhill side in a cooperative manner. However, by mutation, we could destabilize the 尾-finger further and change the rate-limiting step such that an initial conformational change becomes rate limiting. This switch in rate-limiting step shows that multistep binding mechanisms are likely to be found among flexible and intrinsically disordered regions of proteins.