Backbone Effects on Protein Stability and Folding

主链对蛋白质稳定性和折叠的影响

• 批准号: 7031476
• 负责人: PHILIP E DAWSON
• 金额: $ 33.74万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7031476
• 关键词: X ray crystallography; analytical ultracentrifugation; chemical kinetics; chymotrypsin inhibitor; circular dichroism; combinatorial chemistry; fluorescence spectrometry; matrix assisted laser desorption ionization; peptide chemical synthesis; protein engineering; protein folding; protein sequence; protein structure function; stop flow technique; thermodynamics; thermostability

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.

描述（由申请人提供）：本研究计划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使用定点突变的氨基酸侧链的作用上。我们建议偏离这一趋势，通过使用蛋白质的全合成来化学修饰多肽骨架。我们相信，骨架的系统性变化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，α-羟基酸可以纳入蛋白质的位点特异性的方式使用肽合成和化学连接方法。我们已经利用这种修改来分析GCN 4卷曲螺旋中的特定氢键的能量贡献，并在动力学研究中，胰凝乳蛋白酶抑制剂CI 2的折叠过渡态。这些研究表明，骨架修饰提供了直接的信息，在自然状态和折叠过渡态系综，没有观察到使用传统的侧链诱变方法的骨架氢键的形成。该提案旨在回答有关GCN 4和CI 2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。这些蛋白质已被选择，以利用以前的工作，使用定点诱变来分析这些蛋白质的折叠转换。此外，最近对这些蛋白质的计算分析已经对传统实验诱变策略无法解决的折叠进行了具体预测。我们认为，这种使用非编码的修改，如酯键的热力学和动力学测量的蛋白质折叠，将使新的见解的分子基础的蛋白质折叠和稳定性，并提供数据，为继续发展的计算方法来解决这个问题。