1998 Fiscal Year Final Research Report Summary
NMR Analysis of Pressure-Induced Structural Changes in Prioteins
| Project/Area Number |
09480177
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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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 | KOBE UNIVERSITY |
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Principal Investigator |
AKASAKA Kazuyuki Kobe University, Graduate School of Science and Technology, Professor, 大学院・自然科学研究科, 教授 (50025368)
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| Co-Investigator(Kenkyū-buntansha) |
TAMURA Atuo Kobe, University Graduate School of Science and Technology Lecturer, 大学院・自然科学研究科, 講師 (90273797)
YAMADA Hiroaki Kobe Univdersity, Faculty of Sciences, Asssociate Professor, 理学部, 助教授 (90030767)
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| Project Period (FY) |
1997 – 1998
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| Keywords | high pressure NMR / protein structure / 15N chemical shift / hydrogen bond / conformational change / NOE / BPTI / local unfolding |
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| Research Abstract |
1. We utilized a novel high pressure NMR technique that was developed in our laboratory to study the effect of pressure on protein structures in solution. The technique works on proteins in solution at any pressure between 1 and 2000 bar on a modern high field NMR spectrometer operating at 17.6 T or 750 MHz for 1H.
2. We applied this technique to a number of proteins including 15N -uniformly labeled BPTI (basic pancreatic trypsin inhibitor), helix (1-36) of Bacteriorhodopsin, HPr (histidine-containing phospho-carrier protein), RalGEF-RBD (Ral guanine-nucleotide exchange factor-Ras binding domain). Pressure-induced 1H and 15N chemical shift changes revealed in 15N /1H two-dimensional spectra of peptide NH groups showed that the entire backbone structure of a folded protein responds to pressure, but that the response is non-uniform over the sequence.
3. From pressure-induced chemical shifts, we conclude (1) Distances of practically all the hydrogen bonds of amide NH groups, either with carbonyls or solvent water, are shortened by pressure ; (2) Pressure-induced 15N shifts report non-uniform conformational changes in the polypeptide backbone by pressure.
4. From pressure-induced changes in NOE (Nuclear Overhauser Effect) intensities for BPTI, we conclude that pressure induces selective compactions of certain regions of the protein, with concomitant slowing down of flip-flop motions of some aromatic rings.
5. Finally, from signal intensity measurements in RalGEF-RBD, we found that pressure induces local unfolding of the protein preferentially in loop regions above 1000 bar, followed by unfolding of the entire protein at 2000 bar.
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