An Experimental Strategy to Evaluate the Thermodynamic Stability of Highly Dynamic Binding Sites in Proteins Using Hydrogen Exchange

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• 作者：Josephine C. Ferreon ; James B. Hamburger ; and Vincent J. Hilser
• 刊名：Journal of the American Chemical Society
• 出版年：2004
• 出版时间：October 13, 2004
• 年：2004
• 卷：126
• 期：40
• 页码：12774 - 12775
• 全文大小：56K
• 年卷期：v.126,no.40(October 13, 2004)
• ISSN：1520-5126

文摘

Quantitative characterization of the stability of highly dynamic regions in proteins is a significant goal because it represents a cornerstone to an understanding of the role of dynamics in function. Due to experimental constraints, however, monitoring the local stability of highly dynamic regions using standard hydrogen exchange (HX) methods is not a viable approach. Here, an experimental strategy is outlined that takes advantage of the coupling between stability as monitored by HX and binding affinity as monitored by isothermal titration calorimetry. It is shown that the stability of dynamic regions, which are part of binding sites, can be inferred from the response of the system to Gly mutations at surface-exposed sites. When applied to the analysis of the highly dynamic RT loop of SEM5 C-terminal SH3 domain, this approach reveals that the energetic consequences of the observed conformational heterogeneity are significant.