Understanding protein hydrogen bond formation with kinetic H/D amide isotope effects

被引:69
作者
Krantz, BA
Srivastava, AK
Nauli, S
Baker, D
Sauer, RT
Sosnick, TR
机构
[1] Univ Chicago, Dept Biochem & Mol Biol, Chicago, IL 60637 USA
[2] MIT, Dept Biol, Cambridge, MA 02139 USA
[3] Univ Washington, Howard Hughes Med Inst, Seattle, WA 98195 USA
[4] Univ Chicago, Inst Biophys Dynam, Chicago, IL 60637 USA
基金
美国国家卫生研究院;
关键词
D O I
10.1038/nsb794
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Through the development of a procedure to measure when hydrogen bonds form under two-state folding conditions, alpha-helices have been determined to form proportionally to denaturant-sensitive surface area buried in the transition state. Previous experiments assessing H/D isotope effects are applied to various model proteins, including lambda and Arc repressor variants, a coiled coil domain, cytochrome c, colicin immunity protein 7, proteins L and G, acylphosphatase, chymotrypsin inhibitor II and a Src SH3 domain. The change in free energy accompanied by backbone deuteration is highly correlated to secondary structure composition when hydrogen bonds are divided into two classes. The number of helical hydrogen bonds correlates with an average equilibrium isotope effect of 8.6 +/- 0.9 cal mol(-1) site(-1). However, beta-sheet and long-range hydrogen bonds have little isotope effect. The kinetic isotope effects support our hypothesis that, for helical proteins, hydrophobic association cannot be separated from helix formation in the transition state. Therefore, folding models that describe an incremental build-up of structure in which hydrophobic burial and hydrogen bond formation occur commensurately are more consistent with the data than are models that posit the extensive formation of one quantity before the other.
引用
收藏
页码:458 / 463
页数:6
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