Backbone- Backbone H-Bonds in Protein Folding

描述(申请人提供)：在折叠蛋白质中，含有主链C=O和NH基团的氢键构成了大量的天然接触。然而，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。这在很大程度上是因为蛋白质中的骨架突变很难通过传统的定点突变方案引入。在这里，我们提出了利用全化学合成策略进行定点突变实验，以研究主干-主干氢键在几个模型蛋白质系统的折叠和稳定性中的作用，这些模型蛋白质系统包括：P22 Arc抑制物、4-草酰基转移酶(40T)、CopG和蛋白质L。我们的实验将涉及一系列不同的40T、Arc抑制物、CopG和蛋白质L类似物的全化学合成和生物物理性质，这些类似物在多肽链的特定位置含有酰胺到酯键的突变。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性。这项工作的结果将被用来检验关于主链-主链氢键在蛋白质折叠反应中的基础作用的五个假说。我们将确定：(1)蛋白质中所有骨架-骨架氢键的稳定效果是否相同；(2)位于不同蛋白质结构相似区域(即在a-螺旋中间或f3-折叠末端)的骨架-骨架氢键的稳定效果是否相同；(3)具有相同骨架拓扑但不同氨基酸序列(即-lt；25%序列同源性)的蛋白质的结构等价骨架-骨架氢键的稳定效果是否相同；(4)如果骨架-骨架氢键的稳定作用是对处于蛋白质天然状态的蛋白质折叠中间体的稳定作用；以及(5)骨架-骨架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.