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Experimental Approach to Understanding the Principle of Protein Folding

了解蛋白质折叠原理的实验方法

基本信息

批准号：
01044087
负责人：
YUTANI Katsuhide
金额：
$ 2.75万
依托单位：
Institute for Protein Research, Oska University
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1991
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01044087/
关键词：
Calorimetry Conformational Stability Protein folding Proline Lysozyme Tryptophan Synthase Stability Protein Folding Trypyophan Synthase Calorimetry Proline Mutant Protein Denaturation

项目摘要

We have studied mutant proteins in order to elucicate the role of amino acid residues in protein folding, protein stability, and enzymatic function. In this report, we describe the outline of two papers.1. In order to elucidate the role of a proline residue in protein folding, the unfolding and refolding kinetics of six proline mutants of the human lysozyme (h-lysozyme) were carried out and compared to that of the wild type protein. Our results show that the slow refolding phase observed in the h-lysozyme refolding kinetics cannot be ascribed to proline isomerization reactions. The h-lysozyme contains two proline residues at positions 71 and 103. The refolding kinetics of the P71G/PI03G mutant were found to be similar to those of the wild type protein. Other mutants such as P103G or P71G, and A47P with its three prolines, gave identical slow refolding phases.2. To understand how the alpha and beta_2 subunits of tryptophan synthase from Escherichia coli interact to form an alpha_2beta_2 complex and undergo mutual activation, we have investigated alpha subunits with single amino acid replacements at conserved proline residues. Although the activities of alpha_2beta_2 complexes that contain wild type alpha subunits or alpha subunits substituted at positions 28, 62, 96, and 207 are similar, the activities of alpha_2beta_2 complexes that contain alpha subunits substituted at positions 57 and 132 are remarkably altered. Isothermal calorimetric titrations of wild type beta_2 subunit with wild type alpha subunit and a mutant alpha subunit containing a substitution of glycine for proline at position 132 show that both the affinity and the exothermic association enthalpy are greatly reduced in the mutant alpha subunit although the stoichiometry of association is unchanged. We conclude that proline 132 plays a critical role in subunit interaction and in mutual subunit activation.

我们研究了突变蛋白质，以阐明氨基酸残基在蛋白质折叠、蛋白质稳定性和酶功能中的作用。在这份报告中，我们描述了两个文件的大纲。为了阐明脯氨酸残基在蛋白质折叠中的作用，对人溶菌酶（h-溶菌酶）的6个脯氨酸突变体的去折叠和重折叠动力学进行了研究，并与野生型蛋白质进行了比较。我们的研究结果表明，在h-溶菌酶复性动力学中观察到的缓慢复性阶段不能归因于脯氨酸异构化反应。h-溶菌酶在位置71和103处含有两个脯氨酸残基。P71 G/PI 03 G突变体的复性动力学与野生型蛋白相似。其他突变体如P103 G、P71 G和A47 P及其三个脯氨酸，也表现出相同的缓慢复性相.为了了解大肠杆菌色氨酸合酶的α和β_2亚基如何相互作用形成α_2 β_2复合物并相互激活，我们研究了在保守脯氨酸残基处具有单个氨基酸取代的α亚基。尽管含有野生型α亚基或在位置28、62、96和207处被取代的α亚基的α_2 β_2复合物的活性相似，但含有在位置57和132处被取代的α亚基的α_2 β_2复合物的活性显著改变。对野生型β_2亚基、野生型α亚基和132位脯氨酸被甘氨酸取代的突变型α亚基的等温量热滴定表明，突变型α亚基的亲和力和放热缔合焓都大大降低，但缔合的化学计量比不变。我们得出结论，脯氨酸132在亚基相互作用和相互亚基激活中起着至关重要的作用。