Backbone Effects on Protein Stability and Folding

这项研究计划的主要目标是了解分子之间的关系， 多肽链的结构及其折叠成确定的三维结构的能力。大多数研究 关于蛋白质折叠和稳定性的研究主要集中在氨基酸侧链的作用上， 诱变我们建议偏离这一趋势，通过使用蛋白质的全合成， 修饰多肽骨架。我们相信，脊柱的系统性变异将使我们了解 稳定蛋白质的基本力量以及它们折叠的过程。我们有 证明了α-羟基酸可以以位点特异性的方式掺入蛋白质中， 肽合成和化学连接方法。我们已经利用这种修改来分析能量 GCN 4卷曲螺旋中特定氢键的贡献，以及动力学研究中的折叠转变 胰凝乳蛋白酶抑制剂CI 2的状态。这些研究表明，骨架修饰提供了直接的 关于在天然状态和折叠过渡状态下骨架氢键形成的信息 这是使用传统侧链诱变方法未观察到的系综。 该提案旨在回答关于GCN 4和Cl 2的折叠过渡态的具体问题， 将这些研究扩展到蛋白L和酰基磷酸酶的B1结构域。这些蛋白质 选择利用以前的工作，使用定点诱变来分析折叠 这些蛋白质的转变。此外，最近对这些蛋白质的计算分析已经使特定的 关于折叠的预测不能通过传统的实验诱变策略来解决。我们 我觉得这种使用非编码修饰如酯键的热力学和动力学 蛋白质折叠的测量将使人们对蛋白质折叠的分子基础有新的认识， 稳定性，并提供数据的计算方法，这个问题的持续发展。

The broad objective of this research program is to understand the relationship between the molecular structure of a polypeptide chain and its ability to fold into a defined, three-dimensional structure. Most studies on protein folding and stability have focused on the role of amino acid side-chains using site-directed mutagenesis. We propose to diverge from this trend by using the total synthesis of proteins to chemically modify the polypeptide backbone. We believe that systematic variation of the backbone will give insight into the fundamental forces that stabilize proteins and the processes through which they fold. We have demonstrated thatjpha-hydroxy acids can be incorporated into proteins in a site-specific manner using peptide synthesis and chemical ligation methods. We have utilized this modification to analyze the energetic contributions of specific hydrogen bonds in the GCN4 coiled coil and, in kinetic studies, the folding transition state of the chymotrypsin inhibitor CI2. These studies indicate that backbone modification provides direct information on the formation of backbone hydrogen bonding in the native state and folding transition state ensemble that is not observed using traditional side chain mutagenesis methods. This proposal aims to answer specific questions regarding the folding transition states of GCN4 and CI2 and to extend these studies to the B1 domain of Protein L and acylphosphatase. These proteins have been selected to take advantage of previous work using site directed mutagenesis to analyze the folding transitions of these proteins. In addition, recent computational analyses of these proteins have made specific predictions about folding that cannot be addressed by traditional experimental mutagenesis strategies. We feel that this use of non-coded modifications such as ester bonds for thermodynamic and kinetic measurements of protein folding will enable new insights into the molecular basis of protein folding and stability and provide data for the continued development of computational approaches to this problem.