Structural studies on the chemically and genetically engineered protein with high pressure as a denaturant

高压变性剂化学和基因工程蛋白质的结构研究

• 批准号: 08680652
• 负责人: UENO Hiroshi
• 金额: $ 1.6万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08680652/
• 关键词: Folding; Ribonuclease; Pressure; Protein; Genetic engineering; Denaturation

Amino acid components in pancreatic ribonuclease A and yeast carboxypeptidase Y were investigated for their contribution to the protein stability. Proteins were denatured by heat, high pressure, and chemical denaturants, such as urea and guanidine hydrochloride and the process of denaturation was observed by spectroscopic methods. In order to elucidate the exact roles of amino acid residues, mutant enzymes with amino acid substitution by site-directed mutagenesis were prepared as well as chemically modified ones.The role of phenylalanine was difficult to assign because of its inertness toward chemical modification. Site-directed mutagenesis of Phe120 in ribonuclease A was carried out. This residue locates near active site histidine residues and has been shown its participation to the structural stability.Cold temperature denaturation was observed on carboxypeptidase Y only when high pressure was applied. Detailed molecular mechanism of the cold denaturation process was studied by perturbing the denaturation process withreducing agents, polyols, or salts. Process of irreversibility was also investigated.We have demonstrated that pressure can induce protein structural change. In particular, it becomes a denaturing element when temperature being raised or lowered. Therefore, we believe, pressure should be considered as a factor for the study of protein denaturation.

研究了胰腺核糖核酸酶A和酵母羧肽酶Y中氨基酸组分对蛋白质稳定性的影响。用热、高压和化学变性剂（如尿素和盐酸胍）对蛋白质进行变性，并用光谱法观察变性过程。为了阐明氨基酸残基的确切作用，制备了位点诱变取代氨基酸的突变酶以及化学修饰的突变酶。由于苯丙氨酸对化学修饰的惰性，它的作用难以确定。在核糖核酸酶A中进行了Phe120的定点诱变。该残基位于活性位点组氨酸残基附近，并已证明其参与结构稳定性。羧基肽酶Y只有在高压作用下才发生低温变性。通过用还原剂、多元醇或盐扰动变性过程，详细研究了冷变性过程的分子机理。对不可逆性过程进行了研究。我们已经证明压力可以诱导蛋白质结构改变。特别是，当温度升高或降低时，它成为变性元素。因此，我们认为压力是研究蛋白质变性的一个重要因素。