RAMAN INVESTIGATIONS OF THE MOLECULAR BASIS OF VISION

视觉分子基础的拉曼研究

基本信息

批准号：
3483931
负责人：
RICHARD A MATHIES
金额：
$ 13.84万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
1977
资助国家：
美国
起止时间：
1977-09-01 至 1996-08-31
项目状态：
已结题

项目摘要

Our goal is to understand the molecular mechanism of the photochemical events in visual excitation. We want to understand how rhodopsin shifts the absorption of its 11-cis retinal protonated Schiff base chromophore from 440 nm in solution to 500 nm in rod pigments, and how this absorption maximum is regulated in blue- and red-absorbing pigments. We want to elucidate the mechanism of the 11-cis to all-trans isomerization and of energy storage in the primary photoproduct. We also want to know how this stored energy is used to drive the subsequent kinetic decay processes and protein conformational changes that activate transducin. These goals will be addressed by performing time-resolved and low-temperature resonance Raman experiments to study the structure and kinetics of the chromophore in rhodopsins and through femtosecond (10-15 s) transient absorption spectroscopy. The specific aims are: (1) Resonance Raman spectra will be obtained of rhodopsin site specific mutants at 77 K. By determining which mutations alter the vibrational structure of the chromophore in rhodopsin, we will identify the important residues and interactions that produce the opsin shift. By examining the effect of these mutations on bathorhodopsin, we will identify the residues that are responsible for energy storage. (2) Resonance Raman microscope spectra will be obtained of blue and red absorbing pigments from individual photoreceptor cells at 77 K. These data will enable us to examine the molecular mechanism of the opsin shift in these natural pigments. (3) Femtosecond dynamic absorption spectroscopy with 20 fs time-resolution will be used to determine the time scale for the first step in vision. (4) Picosecond time-resolved resonance Raman spectroscopy will be used to study the structure of the primary photoproducts in vision at room temperature to understand the mechanism of energy storage and to understand the mechanism of the isomerization process. (5) Resonance Raman spectra will be obtained of rhodopsins regenerated with dicis-retinals to probe the ability of the chromophore binding site to accommodate different chromophore geometries and Schiff base orientations. This will help to define the steric and electrostatic/hydrogen-bonding requirements for the formation of a pigment. 6) Finally, time-resolved Raman experiments will be performed on the Microsecond to millisecond time scale to obtain high-quality spectra of the lumi, meta I and meta II intermediates. The structure of the retinal chromophore in each intermediate will be determined and the decay kinetics between these species will be analyzed to determine the kinetic and thermodynamic properties of these transitions.

我们的目标是了解光化学的分子机制视觉激励中的事件。我们想了解视紫红质的变化从中吸收其11盘视网膜质子化的Schiff基本发色团在杆色素中对500 nm的溶液中的440 nm，以及这种吸收如何最大化在蓝色和红色吸收色素中受到调节。我们想阐明11-CIS的机制对全频率异构化和一级光产物中的储能。我们也想知道如何存储的能量用于驱动随后的动力学衰减过程和蛋白质构象变化激活转霉素。这些目标将通过执行时间分辨和低温共振来解决拉曼实验研究发色团的结构和动力学在Rhodopsins中以及通过飞秒（10-15 s）瞬时吸收光谱法。具体目的是：（1）共振拉曼光谱将是通过确定哪个突变改变了视紫红质发色团的振动结构，我们将确定产生的重要残基和相互作用 Opsin Shift。通过检查这些突变对沐浴的影响，我们将确定负责储能的残留物。（2）共振拉曼显微镜光谱将获得蓝色和红色从77 K处吸收来自单个感光细胞的色素。这些数据将使我们能够检查Opsin转移的分子机制这些天然色素。（3）飞秒动态吸收光谱使用20 fs的时间分辨率将用于确定视觉的第一步。（4）Picsecond时期分辨的共振拉曼光谱法将用于研究主要的结构室温下视觉中的光产物，以了解储能并了解异构化的机制过程。（5）将获得Rhodopsins的共振拉曼光谱用DICIS-视网膜再生以探测发色团的能力结合位点可容纳不同的发色团几何形状和席夫基础方向。这将有助于定义空间和形成色素的静电/氢键要求。 6）最后，将对时间分辨的拉曼实验进行微秒至毫秒的时间尺度以获得高质量的光谱 Lumi，Meta I和Meta II中间体。视网膜的结构每个中间体中的发色团将确定并衰减动力学将分析这些物种之间以确定动力学和这些过渡的热力学特性。