Photobiology of Rhodopsin & Bacteriorhodopsin

描述(申请人提供)：本研究的长期目标 项目是了解蛋白质光化学的分子细节 细菌视紫红质与视觉的调节和波长调节 视杆和视锥的色素。我们在目前的授权期内的重点是 研究蓝色、紫色和紫外线锥体，因为人们对它们的了解相对较少 这些颜料。将使用的主要光谱工具包括 单光子和双光子光谱、傅里叶变换红外 光谱学、拉曼光谱、圆二色光谱和脉冲激光 光量热法。将进行的主要生化研究包括 定点突变和生色团类似物替换以及 随机诱变，然后进行波长和光化学筛选 属性。理论研究将严重依赖于MNDOPSDCI的使用 用分子轨道理论探讨分子的光物理性质 蛋白质结合的发色团。这一赠款期间的一个重要和新的目标是 在MNDOPSDCI中加入圆二色谱的预测 程序，直接目标是使用实验CD光谱来分析 视紫红质和视锥细胞色素的蛋白结合部位。我们还将使用 考察基态的从头算和半经验分子轨道理论 用分子力学研究生色团结合部位的性质 描述蛋白质的其余部分。我们的目标是联合 协同计划中的实验和理论，两者都得到了加强。此外 对于上面概述的更具全球性的目标，我们将寻求回答以下问题 具体问题：(1)波长的主要机制是什么 锥体色素的调节？(2)是什么导致了显著的 细菌视紫红质与感官物质吸收光谱的差异 视紫红质II？(3)氯结合的机制是什么？ 圆锥体颜料中的位置影响光谱性质 长波长锥体颜料中的生色团？(4)钙在哪里？ 细菌视紫红质中的结合位点，以及这些结合位点如何影响 结合发色团的光物理性质？(5)具体是什么 蛋白生色团的相互作用决定了6-S-顺式与 6-S-反式环结合发色团的构象及视紫红质为什么 选择6-S顺式，细菌视紫红质和感觉视紫红质选择 6-S-反式构象？

DESCRIPTION (provided by applicant): The long-term objectives of this research program are to understand the molecular details of protein photochemical mediation and wavelength regulation in bacteriorhodopsin and the visual pigments of rods and cones. Our emphasis for the present grant period is to study the blue, violet and uv cones because relatively little is known about these pigments. The key spectroscopic tools that will be used include one-photon and two-photon spectroscopy, Fourier-transform infrared spectroscopy, Raman spectroscopy, CD spectroscopy and pulsed laser photocalorimetry. The principal biochemical studies to be undertaken include site-directed mutagenesis and chromophore analog substitutions as well as random mutagenesis followed by screening for wavelength and photochemical properties. The theoretical studies will rely heavily on the use of MNDOPSDCI molecular orbital theory to probe the photophysical properties of the protein-bound chromophores. An important and new goal of this grant period is to add the prediction of circular dichroism spectra to the MNDOPSDCI procedures, with the immediate goal of using experimental CD spectra to analyze the protein binding sites of rhodopsin and the cone pigments. We will also use ab-initio and semiempirical molecularorbital theory to examine the ground state properties of the chromophore binding sites while using molecular mechanics to describe the remaining portions of the protein. Our goal is to combine experiment and theory in a synergistic program that enhances both. In addition to the more global goals outlined above, we will seek to answer the following specific questions: (1) What are the principal mechanisms of wavelength regulation in the cone pigments? (2) What is responsible for the significant difference in the absorption spectra of bacteriorhodopsin versus sensory rhodopsin II? (3) What are the mechanisms through which the chloride binding sites in the cone pigments influence the spectroscopic properties of the chromophores in the long wavelength cone pigments? (4) Where are the calcium binding sites in bacteriorhodopsin, and how do these sites influence the photophysical properties of the bound chromophore? (5) What specific proteinchromophore interactions are responsible for selecting 6-s-cis versus 6-s-trans ring conformations of the bound chromophore, and why does rhodopsin select the 6-s-cis while bacteriorhodopsin and sensory rhodopsin select the 6-s-trans conformation?