| Project/Area Number |
07680725
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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 |
Biophysics
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| Research Institution | Kyushu University |
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Principal Investigator |
UEDA Tadashi Kyushu Univ., Grad.Sch.Pharm.Sci., Assoc.Prof., 大学院・薬学研究科, 助教授 (90184928)
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| Co-Investigator(Kenkyū-buntansha) |
KAWANO Keiichi Hokkaido Univ., Grad.Sch.Pharm.Sci., Assoc.Prof., 大学院・理学研究科, 助教授 (10136492)
IMOTO Taiji Kyushu Univ., Grad.Sch.Pharm.Sci., Prof., 大学院・薬学研究科, 教授 (90038282)
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| Project Period (FY) |
1995 – 1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1996: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1995: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Keywords | lysozyme / NMR / order parameter / fluctuation / ^<15>N labeling / activity / NMR(核磁気共鳴法) / 安定同位体ラベル化 / Pichia / 核磁気共鳴法(NMR) / 大腸菌 / ゆらぎ |
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| Research Abstract |
Recently, we have shown that mutant hen lysozyme where Arg14 and His15 are simultaneously deleted has a higher activity against glycol chitin than that of wild lysozyme while mutated region is far from the active site cleft. From analysis of H-D exchange rate constants in Trp residues of the wild and the mutant lysozyme using NMR,the exchange rate constants in Trp63 of mutant lysozyme, which is located far from the mutated site, was larger than that of wild lysozyme. These results indicated that the increased activity depended on the increased fluctuation (Imoto et al., 1994, Protein Engng.).
Therefore, in this research, in order elucidate whether the increased activity was resulted from the local fluctuation in the active site cleft or the global one of whole molecule, we prepared ^<15>N uniformly labeled wild and mutant lysozymes from Pichia pastoris and measured the relaxation time (T_1 and T_2) of nitrogen atoms and NOEs between ^1H and ^<15>N in them in the presence or absence of substrate analogue. Order parameters in every residues of ^<15>N uniformly labeled wild and mutant lysozymes were calculated by model free analysis of the relaxation time (T_1 and T_2) of nitrogen atoms and NOEs between ^1H and ^<15>N.As the results, it was elucidated that almost all of the order parameters in the mutant lysozyme were smaller than those of wild lysozyme, resulting in that the increased activity was resulted from the global fluctuation of whole molecule.
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