Backbone Effects on Protein Stability and Folding

描述(由申请人提供)：该研究计划的广泛目标是了解多肽链的分子结构与其折叠成定义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定点突变技术研究氨基酸侧链的作用。我们建议通过使用蛋白质的总合成来对多肽骨架进行化学修饰来偏离这一趋势。我们相信，骨架的系统变化将使我们深入了解稳定蛋白质的基本力量以及它们折叠的过程。我们已经证明，利用多肽合成和化学连接的方法，α-羟基酸可以以一种特定的方式结合到蛋白质中。我们利用这一修饰分析了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凝乳酶抑制剂CI2的折叠过渡态。这些研究表明，骨架修饰提供了关于自然态和折叠过渡态系综形成骨架氢键的直接信息，这是传统的侧链突变方法所没有观察到的。这项建议旨在回答有关GCN4和CI2折叠过渡态的具体问题，并将这些研究扩展到蛋白质L和酰基磷酸酶的B1结构域。这些蛋白质被选择来利用以前的工作，使用定点突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对折叠做出了特定的预测，这是传统的实验性突变策略无法解决的。我们认为，这种非编码修饰的使用，如酯键用于蛋白质折叠的热力学和动力学测量，将使人们能够对蛋白质折叠和稳定性的分子基础有新的见解，并为继续发展计算方法来解决这个问题提供数据。

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.