RAMAN INVESTIGATIONS OF THE MOLECULAR BASIS OF VISION

视觉分子基础的拉曼研究

• 批准号: 3256448
• 负责人: RICHARD A MATHIES
• 金额: $ 7.37万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-09-01 至 1991-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3256448
• 关键词: Raman spectrometry; Schiff bases; bioenergetics; chromophore; cis trans isomerization; conformation; fresh water environment; nuclear magnetic resonance spectroscopy; retinaldehyde; rhodopsin; rod cell; visual photoreceptor

Our long-term goal is to determine the molecular mechanism for the photochemical events in visual excitation. We want to understand how rhodopsin shifts the absorption maximum of its 11-cis retinal protonated Schiff base chromophore from 440 nm to 500 nm in rod pigments, and how this interaction is altered in blue and red cone pigments. The mechanism of the 11-cis yield 11-trans photoisomerization and of energy storage in the primary photoproduct, bathorhodopsin, will also be determined. These goals will be addressed by using resonance Raman scattering to obtain vibrational spectra of the protein bound chromophore. Vibrational analyses will then be used to determine chromophore structure. The specific aims are: 1) Resonance Raman spectra will be obtained of rhodopsin, isorhodopsin and bathorhodopsin pigments whose retinal chromophores have been labeled with 13C and 2H. Isotopic derivatives will also be used to assign the vibrations of the 11-cis, 9-cis and all-trans retinal Schiff base model compounds. Comparison of these frequencies and assignments will allow us to determine the "opsin shift" of the C-C and C=C modes. These empirical opsin shifts should allow us to pinpoint the location(s) of the opsin perturbations responsible for Lambda max-regulation and energy storage. 2) Vibrational force field calculations will be performed and protein-induced changes in the force constants, normal modes, and Raman intensities will be analyzed to provide more quantitative information on the nature of these protein perturbations. 3) Magic angle sample spinning 13C-NMR spectra will be obtained of rhodopsin and isorhodopsin regenerated with specific 13C-labeled retinals. The chemical shifts, relaxation times, and tensor elements will be used as an additional probe of chromophore structure and chromophore-protein interactions. 4) Models for chromophore structure and protein-chromophore interactions in rhodopsin and bathorhodopsin will be developed and evaluated with QCFF-Pi calculations. 5) Resonance Raman microscopy will be used to study the mechanism(s) of Lambda max-regulation in a variety of rod and cone photoreceptors to test the generality of the ideas developed in (1)-(4). 6) In a related project, resonance Raman spectra of retinoid binding proteins will be examined to determine the structure of the bound retinal chromophore. This work will tell us how these proteins solubilize retinoids for transport to and from the retinal rod cell, and should provide additional information on how protein-chromophore interactions produce opsin shifts.

我们的长期目标是确定 视觉激发中的光化学事件。 我们想了解如何 视紫红质会移动其11盘视网膜质子化的最大吸收 Schiff基本发色团从440 nm到500 nm的杆颜料，以及如何 相互作用在蓝色和红色锥色素中改变。 机制 11-Cis产生11种TRANS光异构化和储能中的储能 一级光药物，沐浴的淡淡蛋白酶也将确定。 这些目标 将通过使用共振拉曼散射来解决振动 蛋白质结合发色团的光谱。 然后，振动分析将 用于确定发色团结构。 具体目的是：1） 共振拉曼光谱将获得视紫红质，异摩淡鱼和 视网膜发色团已标记的沐浴者色素 13C和2H。 同位素衍生物也将用于分配 11-CIS，9-CIS和全型视网膜席夫基本模型的振动 化合物。 这些频率和作业的比较将使我们 确定C-C和C = C模式的“ Opsin Shift”。 这些经验 OPSIN班次应允许我们查明OPSIN的位置 负责LAMBDA最大调节和能量存储的扰动。 2） 将进行振动力场计算并诱导蛋白质诱导 力常数，正常模式和拉曼强度的变化将是 分析以提供有关这些性质的更多定量信息 蛋白质扰动。 3）魔术角样品旋转13C-NMR光谱将 可获得视紫红质和异摩淡肽的再生 13C标记的视网膜。 化学移动，放松时间和张量 元素将用作发色团结构的附加探针和 发色团 - 蛋白质相互作用。 4）发色团结构的模型和 视紫红质和沐浴的蛋白质 - 肉眼相互作用将是 通过QCFF-PI计算开发和评估。 5）共鸣拉曼 显微镜将用于研究Lambda最大调节的机制 在各种杆和锥形光感受器中测试一般性 （1） - （4）中提出的想法。 6）在一个相关项目中，共鸣拉曼 将检查类类类结合蛋白的光谱，以确定 结合视网膜发色团的结构。 这项工作将告诉我们如何 这些蛋白质溶解性类视黄素往返于视网膜 杆单元，应提供有关如何 蛋白质 - 肉鸡相互作用会产生蛋白酶的变化。