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

研究了胰腺核糖核酸酶A和酵母羧肽酶Y中的氨基酸组成对蛋白质稳定性的影响。用热变性、高压变性和化学变性剂，如尿素和盐酸胍对蛋白质进行变性，并用光谱方法观察变性过程。为了阐明氨基酸残基的确切作用，我们制备了氨基酸定点突变的突变酶和化学修饰的突变酶。由于苯丙氨酸对化学修饰的惰性，其作用很难分配。对核糖核酸酶A中的Phe120进行了定点突变。该残基位于活性中心组氨酸残基附近，参与了结构的稳定。只有在高压条件下，才能观察到羧基肽酶Y的低温变性。通过用还原剂、多元醇或盐对冷变性过程进行扰动，研究了冷变性过程的详细分子机理。我们还研究了蛋白质的不可逆过程，证明了压力可以引起蛋白质结构的改变。特别是，当温度升高或降低时，它会成为变性元素。因此，我们认为，压力应作为蛋白质变性研究的一个因素。

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.