Experimental Approach to Understanding the Principle of Protein Folding

我们对突变蛋白质进行了研究，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。在本报告中，我们描述了两份文件的提纲。为了阐明蛋白质折叠过程中Pro残基的作用，对6个人溶菌酶(h-lysozyme)的Pro突变体进行了去折叠和复性动力学研究，并与野生型蛋白质进行了比较。我们的结果表明，h-溶菌酶复性动力学中观察到的缓慢复性相不能归因于脯氨酸异构化反应。H-溶菌酶在第71和103位含有两个脯氨酸残基。P71G/PI03G突变体的复性动力学与野生型蛋白相似。其他突变体，如P103G或P71G，以及A47P及其三个脯氨酸，都有相同的缓慢复性阶段。为了了解大肠杆菌色氨酸合成酶的α和β_2亚基是如何相互作用形成α_2β_2复合体并相互激活的，我们研究了保守的脯氨酸残基上单一氨基酸取代的α亚基。虽然含有野生型α亚基或28、62、96和207位取代的α亚基的α_2beta_2复合体的活性相似，但含有57和132位取代的α亚基的α_2beta_2复合体的活性发生了显著变化。野生型β_2亚基与野生型α亚基和突变α亚基132位甘氨酸取代的等温量热滴定表明，虽然缔合的化学计量比没有变化，但突变型α亚基的亲和力和结合热都大大降低。我们认为，Pro 132在亚基相互作用和相互亚基激活中起着关键作用。

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.