Joint research on structural fluctuations in proteins-Studied by hydrogen isotope exchange and high pressure NMR
Grant-in-Aid for international Scientific Research
|Allocation Type||Single-year Grants|
|Research Institution||Kobe University|
AKASAKA Kazuyuki Kobe University, Graduate School of Science and Technology, Professor, 大学院・自然科学研究科, 教授 (50025368)
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 University, Faculty of Science, Associate Professor, 理学部, 助教授 (90030767)
WOODWARD Clare K. University of Minesota, Professor, 生物化学科, 教授
CLARE K. Woo ミネソタ大学, 生物化学科, 教授
|Project Period (FY)
1997 – 1998
Completed(Fiscal Year 1998)
|Budget Amount *help
¥5,500,000 (Direct Cost : ¥5,500,000)
Fiscal Year 1998 : ¥3,000,000 (Direct Cost : ¥3,000,000)
Fiscal Year 1997 : ¥2,500,000 (Direct Cost : ¥2,500,000)
|Keywords||high pressure NMR / 15N pressure shift / shortening of hydrogen bond / 13C pressure shift / BPTI / conformational flexibility / 1H pressure shift / 揺らぎ / BPTI / N-15 NMR / 主鎖コンフォメーション / 高圧 / NMR / 牛脾臓トリプシンインヒビター|
The on-line high pressure high resolution NMR system (750 MHz for 1H), which was designed and constructed in Kobe University, was used to investigate the effect of pressure on basic pancreatic trypsin inhibitor (BPTI) in aqueous solution in the pressure range between 1 and 2000 bar.
The 15N-labeled BPTI was prepared by Prof. G.Woodward, Univ.Minnesota, and the 13C-labeled BPTI was prepared by Prof.G.Montelione, Rutgers Univ., while regular BPTI samples were purchased from Sigma Chem. Co.
1. The 15N/1H two-dimensional spectra were measured under pressure, which showed distinctive low field shifts for 1H (average 0.075ppm/2kbar) and 15N signals (average 0.47ppm/2kbar) of amide groups. These shifts reflected shortening of NH---0=C or NH---water hydrogen bonds.
2. A significant variation in 15N pressure shifts among amide nitrogens (0*1.5ppm/2kbar) indicates that 15N pressure shift also reflects changes in phi and psi angles with pressure, giving a measure of site-specific conformational changes of a polypeptide backbone structure by pressure. These changes are apparently larger in helices and loop regions than in b sheet.
3. From proton NOE measurements, it was disclosed that compression occurs non-uniformly within the protein matrix ; compression was found to be large in the enzyme-binding segment of BPTI, suggesting conformational flexibility in this segment.
4. Preliminary results of 15N spin relaxation measurements showed that pressure does not significantly alter the rapid internal motions of BPTT within the range of 2000 bar.
5. Pressure-induced 13C chemical shifts were also measured, the analysis of which is being made.
6. In view of a body of successful experiments stated above, we did not persue the hydrogen exchange experiments in detail.
Research Products (13results)