Kinetic Studies of Protein Folding Using Protein Engineering

利用蛋白质工程进行蛋白质折叠动力学研究

基本信息

批准号：
03453170
负责人：
KUWAJIMA Kunihiro
金额：
$ 4.42万
依托单位：
University of Tokyo (1992-1993)Hokkaido University (1991)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1993
项目状态：
已结题

项目摘要

In this study, staphylococcal nuclease and goat alpha-lactalbumin cloned and expressed by plasmids of Escherichia coli were used to investigate the mechanism of protein folding. Using various site-directed mutants of these proteins, stability of the molten globule state and kinetics of refolding were analyzed. The following results were obtained.(1) In order to investigate effect of proline isomerization on the refolding kinetics, two proline mutants, Pro117Gly and Pro56Ala, of nuclease were constructed. (2) The mutants of nuclease are known to be classified into two classes : Class I mutants stabilize and class II mutants destabilize the molten globule state. The proteins with the class I (Val66Leu, Gly88Val and their double mutant) and class II (Ala90Ser, Ala69Thr and their double mutant) mutations were purified from Escherichia coli that has the respective mutant plasmids. The unfolding transition of each mutant protein was measured by CD spectroscopy in the peptide region. As expected, the secondary structure of the class I mutants is more stable than that of the wild-type protein, while that of the class II mutants is less stable. (3) The mutations (Ala30Ile, Ala30Thr and Thr33Ile) that change the hydrophobicity of the core of the molecule have been introduced in alpha-lactalbumin, and the effect of the mutations on the stability of the molten globule state was investigated by CD spectroscopy. The Ala30Ile and Thr33Ile mutations that increase the core hydrophobicity increase the stability of the molten globule, while the Ala30Thr mutation that decreases the hydrophobicity decreases the stability of the molten globule.

在这项研究中，使用大肠杆菌的质粒克隆并表达葡萄球菌核酸酶和山羊α-乳糖素，以研究蛋白质折叠的机理。使用这些蛋白质的各种位置定向的突变体，分析了熔融球态的稳定性和重折叠的动力学。获得以下结果。（1）为了研究脯氨酸异构化对重折叠动力学的影响，构建了两个核酸酶的脯氨酸突变体，pro117gly和pro56ala，构建了核酸酶。（2）已知核酸酶的突变体分为两类：I类突变体稳定和II类突变体破坏了熔融的球状状态。具有I类（Val66leu，Gly88Val及其双突变体）和II类（ALA90SER，ALA69THR及其双突变体）突变的蛋白质是从具有相应的突变质粒的大肠杆菌中纯化的。每种突变蛋白的展开过渡是通过肽区域中CD光谱法测量的。如预期的那样，I类突变体的二级结构比野生型蛋白的二级结构更稳定，而II类突变体的二级结构较不稳定。（3）CD光谱研究了CD光谱研究研究了改变分子核心疏水性的突变（ALA30IL，ALA30THR和THR33IL），这些突变对分子核心的疏水性已引入，突变对熔融球体状态稳定性的影响。增加核心疏水性的Ala30ile和Thr33ile突变会增加熔融球的稳定性，而降低疏水性的ALA30THR突变会降低熔融球的稳定性。