Backbone- Backbone H-Bonds in Protein Folding

描述（由申请人提供）：涉及骨架C=O和NH基团的氢键构成折叠蛋白质中的大量天然接触。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。这主要是因为蛋白质中的骨架突变难以通过常规定点诱变方案引入。在这里，我们提出了定点诱变实验，利用全化学合成策略，研究骨干骨干氢键的折叠和稳定性的几个模型蛋白质系统，包括：P22弧阻遏物，4-α-巴豆酸互变异构酶（40 T），CopG，和蛋白L的作用。我们的实验将涉及一系列不同的40 T，Arc阻遏物，CopG和蛋白L类似物，在其多肽链的特定位置含有酰胺酯键突变的总化学合成和生物物理特性。酯键突变的目的是调节这些蛋白质系统的多肽链中的特定酰胺键的氢键特征，这项工作的结果将被用来测试五个假设的基本作用的骨干骨干氢键在蛋白质折叠反应。我们将确定：（1）如果蛋白质中所有骨架-骨架氢键的稳定作用相同;（2）如果位于相似区域的骨架-骨架氢键的稳定作用不同蛋白质结构的末端（即在α-螺旋的中间或在f3-折叠的末端）是相同的;（3）如果在具有相同骨架拓扑结构但不同氨基酸序列的蛋白质中，结构等效的骨架-骨架氢键的稳定作用（即<25%序列同源性）相同;（4）如果骨架-骨架氢键蛋白质折叠中间体的稳定作用与蛋白质天然状态的稳定作用相同;和（5）如果骨架-骨架氢键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.