Backbone Effects on Protein Stability and Folding

描述（由申请人提供）：本研究项目的主要目标是了解多肽链的分子结构与其折叠成确定的三维结构的能力之间的关系。大多数关于蛋白质折叠和稳定性的研究都集中在利用位点定向突变的氨基酸侧链的作用上。我们建议通过使用蛋白质的全合成来化学修饰多肽主链来偏离这一趋势。我们相信，骨干结构的系统性变异将使我们深入了解稳定蛋白质的基本力量及其折叠过程。我们已经证明了-羟基酸可以通过肽合成和化学连接方法以特定位点的方式结合到蛋白质中。我们利用这种修饰分析了GCN4卷曲线圈中特定氢键的能量贡献，并在动力学研究中分析了糜凝胰蛋白酶抑制剂CI2的折叠过渡态。这些研究表明，主链修饰提供了在原生态和折叠过渡态系综中形成主链氢键的直接信息，这是传统侧链诱变方法无法观察到的。本提案旨在回答关于GCN4和CI2折叠过渡态的具体问题，并将这些研究扩展到蛋白L和酰基磷酸酶的B1结构域。选择这些蛋白质是为了利用先前的工作，利用位点定向诱变来分析这些蛋白质的折叠转变。此外，最近对这些蛋白质的计算分析已经对折叠做出了特定的预测，这是传统的实验诱变策略无法解决的。我们认为，使用非编码修饰（如酯键）来进行蛋白质折叠的热力学和动力学测量，将使人们对蛋白质折叠和稳定性的分子基础有新的认识，并为解决这一问题的计算方法的持续发展提供数据。

DESCRIPTION (provided by applicant): 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 that alpha-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.