Backbone Effects on Protein Stability and Folding

主链对蛋白质稳定性和折叠的影响

基本信息

批准号：
7534972
负责人：
PHILIP E DAWSON
金额：
$ 29.44万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-05-01 至 2011-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7534972
关键词：
Acylphosphatase Address Amino Acids Aminoisobutyric Acids Biochemical Biological Models Classification Complement Complex Computer Analysis Crystallography Data Development Elements Esters Fluorescence Functional RNA Generations Glycine Human Hydrogen Bonding Hydroxy Acids Individual Kinetics Leucine Zippers Ligation Measurement Measures Methods Modeling Modification Molecular Molecular Conformation Molecular Structure Muscle Mutagenesis Mutation Nature Peptides Phase Phosphoric Monoester Hydrolases Process Property Protein Biosynthesis Protein Engineering Proteins Relative (related person)Research Research Personnel Role Science Series Side Site Site-Directed Mutagenesis Solid Solvents Source System Techniques Tertiary Protein Structure Testing Thermodynamics Variant Vertebral column Work alpha helix analog analytical ultracentrifugation base beta pleated sheet chemical synthesis chymotrypsin inhibitor combinatorial chemistry computer studies insight interest mutant novel peptide chemical synthesis polypeptide programs protein folding protein structure synthetic protein three dimensional structure tool trend

项目摘要

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.

该研究计划的广泛目标是了解分子之间的关系多肽链的结构及其折叠成定义的三维结构的能力。大多数研究关于蛋白质折叠和稳定性的重点是使用位置定向的氨基酸侧链的作用诱变。我们建议通过使用蛋白质的总合成与化学的总合成来与这种趋势不同修改多肽主链。我们认为，骨干的系统变化将洞悉稳定蛋白质及其折叠过程的基本力。我们有证明可以使用位点特异性的方式将羟基酸酸以特异性方式掺入蛋白肽合成和化学连接方法。我们利用这种修改来分析能量在GCN4盘绕线圈和动力学研究中，特定氢键的贡献胰凝乳蛋白酶抑制剂CI2的状态。这些研究表明，骨干修改提供了直接的有关在本地状态和折叠过渡状态形成主链氢键的信息使用传统的侧链诱变方法未观察到的集合。该建议旨在回答有关GCN4和CI2和CI2和将这些研究扩展到蛋白L和酰基磷酸酶的B1结构域。这些蛋白质已经被选为使用定向诱变来分析折叠的先前工作这些蛋白质的过渡。此外，这些蛋白质的最新计算分析已成为特定的传统的实验诱变策略无法解决有关折叠的预测。我们认为这种非编码修饰的使用，例如用于热力学和动力学的酯键蛋白质折叠的测量将使新见解对蛋白质折叠的分子基础和稳定性并为继续开发该问题的计算方法提供数据。