偏光赤外分光を用いたロドプシンの動的構造解析

使用偏振红外光谱分析视紫红质的动态结构

• 批准号: 11480193
• 负责人: KANDORI Hideki
• 金额: $ 9.79万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11480193/
• 关键词: polarized IR; infrared spectroscopy; bacteriorhodopsin; rhodpsin; hydrogen-bond; isotope-effect; mutant; structure changes

This research project aims at revealing the correlation between structure and function in rhodopsins by means of polarized infrared spectroscopy. The target molecules are bacteriorhodopsin (bR) as a light-driven proton pump, halorhodopsin (hR) as a light-driven chloride pump, rhodopsin (Rh) as a light-sensor in twilight vision, and color visual pigments (Chicken-Red and Chicken-Green) as light-sensors in color vision. Infrared spectroscopy of these proteins as well as their mutants and isotope-labeled molecules provided the following results.(1) Bridged water stretching vibrations inside bR under strong hydrogen bond were directly observed by highly refined polarized infrared spectroscopy. Rotation of a water molecule was observed accompanying retinal isomerization.(2) Polarized infrared spectroscopy of bR provides information on the structural changes of protein side upon photoisomerization (K intermediate). Investigation of isotope-labeled sample on threonine side chain and mutants r … More evealed that 3 of the threonines of a total of 18 change their hydrogen bonding. The structural change of Thr17, which is located > 11 Å from the retinal chromophore, implicates a specific perturbation channel in the protein that accompanies the retinal motion.(3) Polarized infrared spectroscopy of bR provides information on the structural changes of protein side before and after the primary proton transfer (L and M intermediates). It was found that hydrogen bond of Thr89 is strengthened upon photoisomerization, and the strong hydrogen bond is persistent even after the "switch" event for proton pump. The results implicate that the Thr89-Asp85 region is not involved in the switch, suggesting that the switch is indeed local machinery.(4) Protein structural changes of hR, Chicken-Red, Chicken-Green, and photoactive yellow protein were extensively studied by low-temperature infrared spectroscopy.(5) Excited-state dynamics of bovine rhodopsin was studied by femtosecond fluorescence spectroscopy. Less

本研究旨在利用偏振红外光谱技术揭示视紫红质的结构与功能之间的相关性。目标分子是作为光驱动质子泵的细菌视紫红质（bR）、作为光驱动氯离子泵的盐视紫红质（hR）、作为微光视觉中的光传感器的视紫红质（Rh）和作为色觉中的光传感器的彩色视色素（鸡红和鸡绿）。这些蛋白质以及它们的突变体和同位素标记的分子的红外光谱提供了以下结果。(1)通过高精细偏振红外光谱直接观察到bR内强氢键作用下的桥水伸缩振动。观察到伴随着视网膜异构化的水分子的旋转。(2)bR的偏振红外光谱提供了光异构化（K中间体）后蛋白质侧结构变化的信息。苏氨酸侧链及突变体r同位素标记样品的研究 ...更多信息 在总共18个苏氨酸中，有3个改变了它们的氢键。Thr 17的结构变化，其位于视网膜发色团> 11 nm处，暗示了伴随视网膜运动的蛋白质中的特定扰动通道。(3)bR的偏振红外光谱提供了关于初级质子转移（L和M中间体）前后蛋白质侧结构变化的信息。结果表明，Thr 89的氢键在光异构化过程中得到了增强，并且这种强氢键在质子泵的“开关”事件后仍然存在。结果暗示Thr 89-Asp 85区域不参与开关，表明开关确实是局部机制。(4)利用低温红外光谱技术研究了hR蛋白、鸡红蛋白、鸡绿蛋白和光敏黄蛋白的结构变化。(5)利用飞秒荧光光谱研究了牛视紫红质的激发态动力学。少

项目成果

Kandori, H., Yamazaki, Y., Shichida, Y., Raap, J., Lugtenburg, J., Belenky, M., and Herzfeld, J.: "Tight Asp85-Thr89 Association during the Pump Switch of Bacteriorhodopsin"Proc.Natl.Acad.Sci.USA. 98. 1571-1576 (2001)

Kandori, H.、Yamazaki, Y.、Shichida, Y.、Raap, J.、Lugtenburg, J.、Belenky, M. 和 Herzfeld, J.：“细菌视紫红质泵开关期间的紧密 Asp85-Thr89 关联”Proc

Kandori, H., Kinoshita, N., Yamazaki, Y., and Shichida, Y.: "FTIR Spectroscopy Reveals Water Structural Changes of Bacteriorhodopsin upon Photoisomerization."in Fourier Transform Spectroscopy (K.Itoh and M.Tasumi, Eds), Waseda University Press. 455-456 (1

Kandori, H.、Kinoshita, N.、Yamazaki, Y. 和 Shichida, Y.：“FTIR 光谱揭示光异构化时细菌视紫红质的水结构变化。”傅里叶变换光谱（K.Itoh 和 M.Tasumi，编辑），

Yamazaki Y.: "Fourier Transform Infrared Studies on Conformation Changes of bd-type Ubiguinol Oxidase from Escherichia coli upon Photoreduction of the Redox Metal Centers"J. Biochem.. 125. 1131-1136 (1999)

Yamazaki Y.：“基于氧化还原金属中心光还原作用的大肠杆菌 bd 型泛甘醇氧化酶构象变化的傅立叶变换红外研究”J.

Kandori,H.: "Local and Distant Protein Structural Changes upon Photosomerization of the Retinal in Bacteriorhodopsin"Proc.Natl.Acad.Sci.USA. 97. 4643-4648 (2000)

Kandori，H.：“细菌视紫红质中视网膜光异构化后的局部和远处蛋白质结构变化”Proc.Natl.Acad.Sci.USA。

Chon, Y.-S., Kandori, H., Sasaki, J., Lanyi, J.K., Needleman, R., and Maeda, A.: "Existence of Two L Photointermediates of Halorhodopsin from Halobacterium salinarum, Different in their Protein and Water FTIR Bands."Biochemistry. 38. 9449-9455 (1999)

Chon, Y.-S.、Kandori, H.、Sasaki, J.、Lanyi, J.K.、Needleman, R. 和 Maeda, A.：“来自盐杆菌的盐视紫红质的两种 L 光中间体的存在，其蛋白质和结构不同