| Project/Area Number |
06680642
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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 | GUNMA UNIVERSITY |
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Principal Investigator |
WAKAMATSU Kaori Gunma University, Faculty of Engineering, Associate Professor, 工学部, 助教授 (40222426)
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| Co-Investigator(Kenkyū-buntansha) |
KOHNO Toshiyuki Mitsubishi Kasei Institute for Life Sciences, Dept.Structure Analysis, Researche, 構造解析研究室, 研究員
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| Project Period (FY) |
1994 – 1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1996: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1995: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1994: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | G protein / peptide / mastoparan-X / expression system / stable isotope labeling / NMR / TRNOE / CD / 重水素化 / レセプター / ペプチド / マストパラン / 発現 |
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| Research Abstract |
We obtained the following results by physicochemical analyzes of G protein-receptor interactions which ultimately aim in elucidating detailed mechanisms of signal transduction via G proteins :
1.Expression of G protein alpha-subunits : We set up a system to express and easily purify recombinant Gilalpha. As high as 50 mg/L culture of Gilalpha could be prepared by Ni^<2+>-affinity chromatography of Gilalpha by attaching a histidine-tag at the N-terminus. Deuterated Gilalpha could also be obtained by this system. Aslo prepared was a K349P mutant which corresponds to the unc mutant in Gsalpha. As expected, this protein was not activated by receptor mimetics. Expression systems for Goalpha and Gsalpha were setup though the latter needs more refinements.
2.Expression and stable isotope labeling of peptides : To analyze interactions between receptor-mimetic peptides and G proteins by NMR in detail, we set up a system that expresses these peptides as a fusion protein with ubiquitin and labels them with stable isotopes such as ^<13>C and ^<15>N.
3.Conformation of Gilalpha-bound mastoparan-X : By TRNOE analysis in the presence of Gilalpha of mastoparan-X uniformly labeled with ^<15>N alone or both ^<13>C and ^<15>N,a precise conformation of mastoparan-X bonud to Gilalpha was obtained with rmsd as small as 0.33*. Residues from the third one to the C-terminus was found to take an alpha-helical conformation.
4.Conformational change of Gilalpha upon activation by receptor mimetics : To investigate the mechanism where by stimulation by a liganded receptor enhances the GDP release from G protein, secondary structure change of Gilalpha caused by receptor mimetics was analyzed by CD.The alpha-helix content of Gilalpha was found to decrease in parallel with its activation.
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