1997 Fiscal Year Final Research Report Summary
Structural studies on the chemically and genetically engineered protein with high pressure as a denaturant
| Project/Area Number |
08680652
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Structural biochemistry
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
UENO Hiroshi Kyoto University, Graduate School of Agriculture Associate Professor, 農学研究科, 助教授 (30241160)
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| Co-Investigator(Kenkyū-buntansha) |
HAYASHI Rikimaru Kyoto University Graduate School of Agriculture Professor, 農学研究科, 教授 (90027186)
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| Project Period (FY) |
1996 – 1997
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| Keywords | Folding / Ribonuclease / Pressure / Protein / Genetic engineering / Denaturation |
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| Research Abstract |
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.
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