Backbone- Backbone H-Bonds in Protein Folding

描述（由申请人提供）：包含主链C=O和NH基团的氢键构成折叠蛋白质中大量的天然接触。然而，相对而言，人们对它们对蛋白质折叠和稳定性的贡献的性质知之甚少。这主要是因为传统的定点诱变方案难以引入蛋白质中的骨干突变。在这里，我们提出了利用全化学合成策略的定点诱变实验，以研究骨干-骨干氢键在几种模型蛋白质系统的折叠和稳定性中的作用，包括：我们的实验将涉及一系列不同的40T、Arc阻遏物、CopG和蛋白L类似物的总化学合成和生物物理表征，这些类似物在其多肽链的特定位置含有酰胺到酯键突变。酯键突变被设计用来调节这些蛋白质系统多肽链中特定酰胺键的氢键特征。这项工作的结果将用于测试关于主-主氢键在蛋白质折叠反应中的基本作用的五个假设。我们将确定：(1)蛋白质中所有主-主氢键的稳定作用是否相同；(2)不同蛋白质结构中位于相似区域（即a-螺旋中间或f3-sheet末端）的主-主氢键的稳定作用是否相同；(3)具有相同主链拓扑结构但不同氨基酸序列（即序列同源性<25%）的蛋白质中，结构等效的主-主氢键的稳定作用是否相同；(4)主-主氢键蛋白质折叠中间体的稳定作用是否优于蛋白质的天然状态；(5)主-主氢键是否有助于蛋白质折叠过渡态的稳定。

DESCRIPTION (provided by applicant): Hydrogen bonds involving backbone C=O and NH groups constitute a large number of the native contacts in folded proteins. However, relatively little is known about the nature of their contribution to protein folding and stability. This is largely because backbone mutations in proteins are difficult to introduce by conventional site-directed mutagenesis protocols. Here we propose site-directed mutagenesis experiments utilizing total chemical synthesis strategies to study the role of backbone-backbone hydrogen bonds in the folding and stability of several model protein systems including: P22 Arc repressor, 4-oxalocrotonate tautomerase (40T), CopG, and protein L. Our experiments will involve the total chemical synthesis and the biophysical characterization of a series of different 40T, Arc repressor, CopG and protein L analogues that contain amide to ester bond mutations at specific locations in their polypeptide chains. The ester bond mutation is designed to modulate the hydrogen bonding characteristics of specific amide bonds in the polypeptide chains of these protein systems.The results of this work will be used to test five hypotheses about the fundamental role of backbone-backbone hydrogen bonds in protein folding reactions. We will determine: (1) if the stabilizing effects of all backbone-backbone hydrogen bonds in proteins are the same; (2) if the stabilizing effects of backbone-backbone hydrogen bonds located in similar regions (i.e. in the middle of an a-helix or at the end of a f3-sheet) of different protein structures are the same; (3) if the stabilizing effects of structurally equivalent backbone-backbone hydrogen bonds in protein's with the same backbone topology but different amino acid sequences (i.e. <25% sequence homology) are the same; (4) if the stabilizing effects of backbone-backbone hydrogen bonds protein folding intermediates are to those in the protein's native state; and (5) if backbone-backbone hydrogen bonds contribute to the stabilization of protein folding transition states.