Dynamic Structural Analyzes of the Photointermediates of Bacteriorhodopsin

细菌视紫红质光中间体的动态结构分析

• 批准号: 05680579
• 负责人: KATAOKA Mikio
• 金额: $ 1.28万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05680579/
• 关键词: Bacteriorhodopsin; Photoreaction Intermediate; Light-driven Proton Pump; X-ray Diffraction; Schiff Base; Amino-acid Substitution; X線回折; 時分割X線回折; 遺伝子操作; 点突然変異体

The major purpose of this researchi is the structural analysis of each photointermediate of bacteriorhodopsin (bR), in order to understand the molecular mechanism of its light-driven proton pump.Despite that D85N mutant bR does not generate the M-intermediate by photo-reaction, alkaline D85N has similar properties to the M-intermediate. The structure of D85N at alkaline pH was compared with the structure at neutral pH.In order to record the X-ray diffraction patterns at the same place of the same oriented specimen under different pH,we developed an ammonium treatment method. The changes in diffraction profiles from neutral to alkaline pH resemble to those from the grand state to the M-intermediate of wild type bR and D96N.The structural changes occurred at alkaline pH are remarkable around helices C,F and G by a difference Fourier synthesis. These structural changes are common for the structural changes in the M intermediate. Although the similar changes were observed for D85N/D96N,the … More extent of the changes were much smaller than that of D85N.X-ray diffraction profile of D85N/D96N at neutral pH clearly indicates that some portion of the double mutant take the M-like structure even at neutral pH.We conclude that the deprotonation of Schiff base stabilizes the M-type structure, and that the M-type structure stimulates the deprotonation of Schiff base. Schiff base proton can interact with the cytoplasmic surface only in the M-type structure, while the proton can interact with the extracellular surface only in the grand state structure.The M-decay processes at various pH were studied by kinetic spectroscopy and kinetic X-ray diffraction. The reversion of the structure from the M-type to grand state is closely correlated with the reprotonation of Schiff base. Using mercury-labeled cysteine substitution mutant bR,we revealed that the mercury position can be identified by X-ray diffraction. This technique will be utilized for the further quantitative description of the structural change in detail. Less

本研究的主要目的是分析细菌视紫红质(BR)的每个光中间体的结构，以了解其光驱动质子泵的分子机制。尽管D85N突变体BR不通过光反应产生M-中间体，但碱性D85N具有与M-中间体相似的性质。比较了D85N在碱性pH和中性pH下的结构，为了记录同一取向样品在不同pH下同一位置的X射线衍射图，我们开发了一种氨化处理方法。从中性到碱性pH的衍射谱变化与野生型BR和D96N从大态到M中间体的变化相似。通过差值傅立叶合成，在碱性pH下螺旋C、F和G附近发生了显著的结构变化。这些结构变化对于M中间体的结构变化是常见的。虽然D85N/D96N也有类似的变化，但…D85N/D96N在中性pH下的X-射线衍射图表明，即使在中性pH下，D85N/D96N的部分突变体仍具有类M结构。我们认为，Schiff碱的去质子化稳定了M型结构，而M型结构促进了Schiff碱的去质子化。希夫碱质子只能在M型结构中与胞质表面相互作用，而质子只能在宏态结构中与胞外表面相互作用。用动力学光谱和动力学X射线衍射研究了不同pH下的M衰变过程。结构从M型向大态的反转与Schiff碱的再质子化密切相关。利用汞标记的半胱氨酸取代突变体BR，我们发现通过X射线衍射可以确定汞的位置。这项技术将被用来进一步详细地定量描述结构变化。较少

项目成果

Mikio Kataoka: "Trimeric mutant bacteriorhodopsin,D85N,shows a monophasic CD spectrum" FEBS Letters. 333. 111-113 (1993)

Mikio Kataoka：“三聚体突变细菌视紫红质，D85N，显示单相 CD 谱” FEBS Letters。

Kenichi Mihara: "Structural studies on the M intermediate with the use of mutant bacteriorhodopsin" Photomedicine and Photobiology. 15. 69-74 (1993)

Kenichi Mihara：“使用突变细菌视紫红质对 M 中间体进行结构研究”光医学和光生物学。

徳永史生: "視覚器一視興奮の分子機構と網膜再生" 生体の化学. 45. 62-70 (1994)

Fumio Tokunaga：“视觉兴奋一目了然和视网膜再生的分子机制”生物化学 45. 62-70 (1994)。

K.Ozaki, M.Ozaki, H.Nagatani & F.Tokunaga: "Maturation of major Drosophila rhodopsin, ninaE,requires chromophore 3-hydroxyretinal" Neuron. 10. 1113-1119 (1993)

K.Ozaki, M.Ozaki, H.Nagatani

Mikio Kataoka: "Structural characterization of molten globule and native states of apomyoglobin by solution X-ray scattering" Journal of Molecular Biology. (印刷中). (1995)

Mikio Kataoka：“通过溶液 X 射线散射对熔球和脱辅基红蛋白的天然状态进行结构表征”，《分子生物学杂志》（1995 年出版）。