Experimental Approach to Understanding the Principle of Protein Folding

为了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，我们研究了突变蛋白。在这篇报告中，我们描述了两篇论文的大纲。为了阐明脯氨酸残基在蛋白质折叠中的作用，对6个人类溶菌酶（h-溶菌酶）脯氨酸突变体的展开和再折叠动力学进行了研究，并与野生型蛋白进行了比较。我们的研究结果表明，h-溶菌酶重折叠动力学中观察到的缓慢重折叠阶段不能归因于脯氨酸异构化反应。h-溶菌酶在71位和103位含有两个脯氨酸残基。发现P71G/PI03G突变体的重折叠动力学与野生型蛋白相似。其他的突变体，如P103G或P71G，以及A47P和它的三个脯氨酸，具有相同的缓慢的再折叠阶段。为了了解大肠杆菌色氨酸合成酶的α亚基和β _2亚基是如何相互作用形成α _2β _2复合物并相互激活的，我们研究了α亚基在保守的脯氨酸残基上替换单个氨基酸的过程。虽然含有野生型α亚基和取代于28、62、96和207位α亚基的α _2beta_2配合物的活性相似，但含有取代于57和132位α亚基的α _2beta_2配合物的活性明显改变。等温量热滴定结果表明，野生型α亚基与突变型α亚基的亲和性和放热缔合焓均显著降低，而α亚基的化学计量量不变。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.