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

1)光活性黄蛋白（PYP） E46Q突变体的原子分辨率结构分析来自嗜盐异硫螺旋藻（Ectothiorhodospira halophila）的PYP是一个分子量为14 KDa的光受体蛋白。发色团对香豆酸通过硫酯键与PYP的Cys69相连。PYP在光吸收过程中经历一个由几种中间体组成的光循环。在基态下，发色团的OH基团被去质子化，由与Glu46和Tyr42的氢键稳定。Gln取代Glu46引起最大吸收红移和pKa的碱移。收集了E46Q突变蛋白晶体的x射线衍射数据，分辨率为1.2 A^^0 （Rmerge=3.7%）。结构的各向异性细化使rrfree降低到0.165/0.191。目前的分析表明，Gln46的氨基基团与发色团形成氢键。详细的结构也解释了吸收光谱和pKa的变化。2)马尾铁氧化还蛋白的高分辨率x射线分析该铁氧化还蛋白是一种电子传递蛋白，具有一个2Fe-2S簇作为活性中心。制备了其晶体，并在SPring-8同步辐射（Rmerge=8.4%）下采集了1.25 A^^0分辨率的衍射数据。用分子置换法确定了其结构。结构细化正在进行中。3)制备用于中子散射的4fe -铁氧还蛋白晶体。对热水解酵母中4fe -铁氧还蛋白进行过表达和纯化。筛选生产适合中子散射的巨型晶体的工作正在进行中。

1) Structure analysis of photoactive yellow protein (PYP) E46Q mutant at atomic resolutionPYP 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 ferredoxinThis 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 scattering4Fe-ferredoxin from B. thermoproteolyticus was overexpressed and purified. Screening to produce giant crystals suitable for neutron scattering is underway.