Functional analysis of proteins based on the tertiary structures focussing on hydrogen atom positions

Research Project

Project/Area Number	14580674
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Single-year Grants
Section	一般
Research Field	Biophysics
Research Institution	Osaka University
Principal Investigator	FUKUYAMA Keiichi FUKUYAMA,Keiichi, 大学院・理学研究科, 教授 (80032283)
Co-Investigator(Kenkyū-buntansha)	NIIMURA Nobuo Ibaraki University, Faculty of Engineering, Professor, 工学部, 教授 (50004453)
Project Period (FY)	2002 – 2003
Project Status	Completed (Fiscal Year 2003)
Budget Amount *help	¥3,500,000 (Direct Cost: ¥3,500,000) Fiscal Year 2003: ¥1,700,000 (Direct Cost: ¥1,700,000) Fiscal Year 2002: ¥1,800,000 (Direct Cost: ¥1,800,000)
Keywords	PYP / Photoreceptor / three-dimentional structure / X-ray analysis / atomic resolution analysis / ferredoxin / ペルオキシダーゼ / 中性子回析 / 精密立体構造 / 水素原子位置
Research Abstract	1) Structure analysis of photoactive yellow protein (PYP) E46Q mutant at atomic resolution PYP from Ectothiorhodospira halophila is a photoreceptor protein with molecular weight of 14 KDa. A chromophore, p-coumaric acid, is linked to Cys69 of PYP by thioester bond. PYP undergoes a photocycle consisting of several intermediates on light absorption. In its groud state OH group of the chromophore is deprotonated, which is stabilized by hydrogen bonds with Glu46 and Tyr42. Replacement of Glu46 by Gln causes red-shift of absorption maximum and alkali-shift of pKa. X-ray diffraction data for E46Q mutant protein crystal have been collected to 1.2 A^^0 resolution (Rmerge=3.7%). Anisotropic refinement of the structure reduced RlRfree to 0.165/0.191. The present analysis has revealed that amindo group of Gln46 is hydrogen bonded to the chromophore. The detailed structure also explained the changes of absorption spectrum and pKa. 2) High resolution X-ray analysis of horsetail ferredoxin This ferredoxin is an electron-transfer protein that has one 2Fe-2S cluster as active center. Its crystals were prepared and the diffraction data were collected to 1.25 A^^0 resolution using synchrotron radiation at SPring-8 (Rmerge=8.4%). The structure was determined by the molecular replacement method. The structure refinement is in progress. 3) Preparation of 4Fe-ferredoxin crystals for neutron scattering 4Fe-ferredoxin from B. thermoproteolyticus was overexpressed and purified. Screening to produce giant crystals suitable for neutron scattering is underway.