PROBING THE FOLDING TRANSITION STATE VIA SIDECHAIN AND BACKBONE MUTATIONS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 主链-主链氢键是β-折叠结构的显著特征。由于它们在这种普遍存在的二级结构中的中心作用，我们将确定骨架-主链H键的形成，特别是β转角形成对β-折叠形成动力学的贡献。这将通过对研究得很好的β-发夹多肽的酰胺到酯的突变体的phi(M)值分析来实现。主链-主链氢键对蛋白质折叠，特别是β-折叠的热力学和动力学的贡献是有争议的。以前，我们使用酰胺到酯的突变来解决这个问题，在这种突变中，主链酰胺被酯取代。酰胺-酯突变是研究蛋白质主链-主链氢键的有力工具，因为(1)酯和酰胺具有相似的键长度和角度；(2)酯和酰胺都具有很高的旋转障碍，并有利于C‘-Oe或C’-NH键的反式构象(“Oe”指酯中的非羰基氧)；以及(3)酰胺-酯突变不会改变突变残基的固有构象倾向或侧链相互作用，因为侧链没有改变。许多研究，包括我们自己在Pin WW结构域上的工作，都表明转弯形成限制了β-发夹和小β-折叠蛋白的折叠速度的假说。为了更好地理解β-折叠的这一基本方面，将通过对β-发夹和/或α-螺旋肽上的酰胺到酯突变的Phi(M)值分析来检验这一假设。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Backbone-backbone H-bonds are prominent features of the structures of beta-sheets. Because of their central role in this ubiquitous secondary structure, we will determine the contribution backbone-backbone H-bond formation in general, and beta-turn formation in particular, to the kinetics of beta-sheet formation. This will be accomplished using a Phi(M)-value analysis of amide-to-ester mutants of a well-studied beta-hairpin peptide. The contribution of backbone-backbone H-bonds to the thermodynamics and kinetics of protein folding in general, and beta-sheet folding in particular, is controversial. Previously, we used amide-to-ester mutations, in which a backbone amide is replaced by an ester, to address this problem. Amide-to-ester mutation is a powerful tool for studying backbone-backbone H-bonding in proteins because (1) esters and amides have similar bond lengths and angles; (2) esters and amides both have a high barrier to rotation and favor a trans conformation about the C'-Oe or C'-NH bond ("Oe" refers to the non-carbonyl oxygen in an ester); and (3) amide-to-ester mutations do not alter the intrinsic conformational propensity or side chain interactions of the mutated residue because the side chain is not altered. Many studies, including our own work on the Pin WW domain, have suggested the hypothesis that turn formation limits the folding rate of beta-hairpins and small beta-sheet proteins. To better understand this fundamental aspect of beta-sheet folding, this hypothesis will be tested by using Phi(M)-value analysis of amide-to-ester mutations on a beta-hairpin and/or alpha-helical peptides.