MOLECULAR MECHANISMS OF LIGHT TRANSDUCTION BY RHODOPSIN

我们研究的长期目标是了解 视紫红质的结构决定了其在视觉转导中的功能。这个 本提案的目的是研究分子机制。 视觉兴奋循环中的一个关键步骤：通过 视紫红质在光吸收后在眼睛中再生。这个 建议的方法是多学科性质的，涉及 光吸收技术组合技术的应用 光谱学、生物化学和分子生物学。再生 机制将在三个模型系统中进行研究：牛和人 (脊椎动物)视紫红质和Rh1果蝇(无脊椎动物)视紫红质。 这些实验旨在解决以下问题： 视紫红质的再生是否通过动力学中间体进行？ 用光谱学可以探测到吗？如果是这样的话，这些有关系吗？ 光吸收产生的光中间体？什么是 脊椎动物和无脊椎动物视紫红质的关键结构差异 这就解释了脊椎动物视紫红质的再生 每个周期需要11顺式视网膜的新鲜分子，但仅 在无脊椎动物身上需要第二个光子？ 具体目标是： 一、确定牛再生途径中的步骤 吸收光谱测定视黄素和11-顺式视网膜中的视紫红质。 研究发现的最常见的视紫红质突变的影响 在视网膜色素变性的再生途径上进行 白花蛇舌草定点突变体的光谱和生化研究 已经在组织培养中表达的牛视紫质。 利用Sf9昆虫细胞表达和纯化Rh1果蝇视紫红质 使用杆状病毒表达系统，并确定 视紫红质和变视紫质之间的光驱动相互转换 表格。 IV.设计并引入牛和Rh1的定点突变 果蝇视紫红质识别负责的关键氨基酸 对于脊椎动物和动物再生途径的差异 无脊椎动物视紫红质。 V.启动实验以获得Rh1的二维晶体 果蝇视紫红质。

The long-term objectives of our research are to understand how the structure of rhodopsin governs its function in visual transduction. The goals of the present proposal are to investigate the molecular mechanisms of a key step in the cycle of visual excitation: the pathway by which rhodopsin is regenerated in the eye following light absorption. The proposed approach is multidisciplinary in nature, and involves the application of a combination of the techniques of optical absorption spectroscopy, biochemistry and molecular biology. Regeneration mechanisms will be studied in three model systems: bovine and human (vertebrate) rhodopsins, and Rh1 Drosophila (invertebrate) rhodopsin. The experiments have been designed to address the following questions: Does the regeneration of rhodopsin proceed through kinetic intermediates detectable by optical spectroscopy? If so, do these bear any relation to the photointermediates generated by light absorption? What are the key structural differences between vertebrate and invertebrate rhodopsins that account for the fact that regeneration of rhodopsin in vertebrates requires a fresh molecule of 11-cis retinal for each cycle, but only requires a second photon in invertebrates? The specific aims are to: I. Determine steps in the pathway of the regeneration of bovine rhodopsin from opsin and 11-cis retinal by absorption spectroscopy. II. Investigate the effect of the most common rhodopsin mutations found in retinitis pigmentosa on the regeneration pathway by carrying out spectroscopic and biochemical studies with site-specific mutants of bovine rhodopsin that have already been expressed in tissue culture. III. Express and purify Rh1 Drosophila rhodopsin from Sf9 insect cells using the baculovirus expression system, and determine steps in the light-driven interconversions between the rhodopsin and metarhodopsin forms. IV. Design and introduce site-specific mutations in bovine and Rh1 Drosophila rhodopsins to identify key amino acids that are responsible for the differences in the regeneration pathways of vertebrate and invertebrate rhodopsins. V. Initiate experiments to obtain two-dimensional crystals of Rh1 Drosophila rhodopsin.