FEMTO/PICOSECOND LASER SPECTROSCOPY OF RHODOPSIN

脊椎动物和无脊椎动物的视觉过程利用反式- 膜蛋白视紫红质(Rh)，用于存储和转导吸收的光 能量。在分子水平上，能量储存/传导机制 涉及(I)单曲的结构中的一系列转换 Rh，视网膜中可见的生色团，以及(Ii)相互作用 视网膜及其蛋白质环境。对于体内的蛋白质来说，这些 过程是脂质构象变化的先决条件，信号 扩增，并最终通过G蛋白转化 (转导蛋白)结合在蛋白质表面，进入神经信号。许多 细菌视紫红质(BR)模拟了Rh机制的某些方面。 比Rh更易于实验研究的光循环。 拟议的研究扩展了该小组在 房间里最早的(飞秒/皮秒)分子过程 考察了温度、Rh光反应。建议中的重点 工作的结构变化与(I)照片和 Batho中间体，大部分吸收的能量最初存储在那里 和(Ii)蓝移中间体(BSI)和光视紫质(LUMI) 它们参与了能量的传递。在这项研究之前， Rh的结构信息仅限于冷冻样品，其中 照片序列被中断，以稳定特定的物种。这个 最近获得的室温Batho的振动光谱 第一次在这个实验室里，展示了低温 振动数据不能准确地描述室温的视觉 机制。 时间分辨相干拉曼技术及其定量分析 测量皮秒所需的非线性光学数据的方法 一种室温蛋白质的结构变化也是最近发生的 在这个实验室里研发的。这些实验克服了 限制前人的Rh光序的不可逆性 努力获取室温数据。人造的(化学的和 同位素修饰的视网膜)和突变的Rh色素(通过 与这些领域的领先研究人员合作)将 以阐明分子运动在特定情况下的重要性 视网膜连接到Rh照片序列。拟议的实验将 表征结构和动力学性质可分配给 Rh光序列中的PHOTO、BASTO、BSI和LUMI中间体。 在实验上，皮秒的成功开发和应用 时间分辨相干反斯托克斯拉曼(PTR/CARS)在这方面开创了先河 实验室将扩大以阐明视网膜如何变化 结构和视网膜-蛋白质的相互作用控制着早期的 Rh照片序列。皮秒瞬时吸收和荧光 测量结果将补充振动光谱学的测量结果。时间- 分辨相干拉曼技术也将扩展到测量偶极子 力矩通过系统沿特定的振动模式变化 偏振控制。还将努力回落 有效地中断这些相应的光反应的反应。 将在BR中进行一些平行研究，以进一步发展和 评估新的实验和分析方法，并扩展我们的 了解这一独立重要的跨膜， 光合作用蛋白质。

Visual processes in vertebrates and invertebrates utilize the trans- membrane protein, rhodopsin (Rh), to store and transduce absorbed light energy. At the molecular level, the energy storage/transduction mechanism involves a series of transformations in (i) the structure of the single visible chromophore in Rh, retinal, and (ii) the interactions between retinal and its protein environment. For an in vivo protein, these processes are prerequisites for lipid conformational changes, signal amplification, and eventually the transformation, via G-protein (transducin) binding at the protein surface, into nerve signals. Many aspects of the Rh mechanisms are mimicked by the bacteriorhodopsin (BR) photocycle which is easier than Rh to study experimentally. The proposed research expands earlier work performed in the group during which the earliest (femto/picosecond) molecular processes in the room temperature, Rh photo-reaction were examined. Emphasis in the proposed work is placed on the structural changes associated with (i) the photo and batho intermediates where much of the absorbed energy is stored initially and (ii) the blue-shifted intermediate (BSI) and lumi-rhodopsin (lumi) which are involved in the energy transduction. Until this study, structural information for Rh has been limited to frozen samples in which the photo-sequence is interrupted to stabilize a particular species. The vibrational spectrum of room-temperature batho, obtained recently for the first time in this laboratory, demonstrated that low-temperature vibrational data do not accurately describe the room-temperature visual mechanism. The time-resolved coherent Raman techniques and quantitative analysis methods of non-linear optical data required to measure picosecond structural changes of a room-temperature protein were also recently developed in this laboratory. These experiments overcome the irreversibility of the Rh photo-sequence which has limited previous efforts to obtain room temperature data. Artificial (chemically- and isotopically-modified retinals) and mutant Rh pigments (provided through collaborations with leading researchers in these respective fields) will be examined to elucidate the importance of molecular motions at specific retinal bonds to the Rh photo-sequence. Proposed experiments will characterize the structures and kinetics properties assignable to the photo, batho, BSI, and lumi intermediates in the Rh photo-sequence. Experimentally, the successful development and application of picosecond time-resolved coherent anti-Stokes Raman (PTR/CARS) pioneered in this laboratory will be expanded to elucidate how changes in the retinal structure and in retinal-protein interactions control the early stages of the Rh photo-sequence. Picosecond transient absorption and fluorescence measurements will complement those of vibrational spectroscopy. The time- resolved coherent Raman techniques also will be expanded to measure dipole moment changes along specific vibrational modes through the systematic control of polarization. Efforts also will be made to measure back reactions which effectively interrupt these respective photo-reactions. Some parallel studies in BR will be undertaken to further develop and evaluate new experimental and analysis methods as well as to expand our understanding of this independently important trans-membrane, photosynthetic protein.