Crystallographic Studies on Protease ー Substrate Interaction Using Genetically - Engineered Inhibitors.
| Project/Area Number |
01480516
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
結晶学
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| Research Institution | Nagaoka University of Technology |
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Principal Investigator |
MITSUI Yukio Faculty of Engineering, Nagaoka University of Technology , Professor, 工学部, 教授 (40012637)
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| Co-Investigator(Kenkyū-buntansha) |
KUMAGAI Izumi Faculty of Engineering , University of Tokyo , Assoc. Professor, 工学部, 助教授 (10161689)
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| Project Period (FY) |
1989 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥4,500,000 (Direct Cost: ¥4,500,000)
Fiscal Year 1990: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1989: ¥3,400,000 (Direct Cost: ¥3,400,000)
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| Keywords | Protease / Protease inhibitor / Protein-protein interaction / Protein engineering / X-ray structure analysis / Crystal structure / Three-dimensional structure of Protein / 蛋白質の立体構造 |
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| Research Abstract |
Crystal structure of free SSI (Streptomyces Subtilisin Inhibitor) and that of SSI complexed with its target enzyme subtilisin BPN' were refined to R-factors of 24% (1.85 A) and 21% (1.8 A) respectively. Furthermore two more related structures were refined in which subtilisin BPN' was complexed with either mutant SSI-1 or mutant SSI-2. In both mutant SSI's, the P1 residue of the inhibitor. Met 73, was converted to Lys through gene manipulation. Additionally, in mutant SSI-2 the P4 residue, Met 70, was converted to Gly.
Comparative examinations of the structural and "thermal" parameters of these structures indicate the following.
(A) Marked rigidification of the SSI structure occurs to some (but not all) parts of SSI upon complex formation with the target enzyme (subtilisin BPN').
(B) The rigidification occurs not only to the "reactive site" segment which is in direct contact with the target enzyme but also to those polypeptide segments which are simply connected to the reactive site through either covalent linkage or van der Waals contacts.
(C) The rigidified polypeptide segments of SSI mentioned above roughly correspond to the segments where marked shifts upon complex formation were observed in C-13 NMR spectra (observed by M.Kainosho and his colleague using various C-13 enriched SSI's).
(D) Basic shape of the SI pocket of subtilisin BPN' seems to be almost fixed irrespective of the nature of its "guest" (P1 residue of the inhibitor).
(E) In contrast, the S4 pocket seems to have intrinsic capacity for induced fit : it is considerably narrowed when its guests, P4 residue has smaller sidechain.
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Report
(3 results)
Research Products
(18 results)
