Single Molecular Biochemistry of The Operating Mechanism of Photoreoeptor G protein and its Trafficking Mechanism.

本研究的主要目的是利用单分子成像技术阐明脊椎动物感光盘膜G蛋白转导蛋白的作用机制。第二个目的是研究G蛋白在视杆细胞内外节之间运输的分子机制。作为第一个任务，PI挑战用近红外荧光化合物（Hilyte 750）标记转导蛋白，并成功。标记的transducin保留其正常的活动，结合光漂白视紫红质，核苷酸交换活性，和PDE 6激活。通过使用这种荧光标记的转导蛋白，Pl通过使用750 nm激光束激发标记物成功地观察到转导蛋白在暗状态下的单分子运动。在暗适应盘膜中，转导蛋白的扩散速度比视紫红质快得多。在可见光照射下，转导素与光漂白的视紫红质形成复合物，该复合物表现出缓慢的光致变色。 关于我们 比视紫红质更扩散。复合物的扩散速率依赖于盘膜的胆固醇含量，即胆固醇结合化合物甲基-β-环糊精的胆固醇消耗加速了转导素-视紫红质复合物的扩散，这表明转导素的激活发生在盘膜中胆固醇依赖性微区的外围。虽然微区的形成机制尚未阐明，但这些结果为我们深入了解脊椎动物光转导系统中G蛋白信号转导机制提供了新的视角。至于转导素在视杆细胞内外节之间的运输，P1还不能将荧光标记的转导素引入视杆细胞外节。然而，Pl的发现是GTP形式的转导蛋白显示在盘膜切口口处的看似微管蛋白纤维的外周点处形成聚集的倾向，这表明转导蛋白与微管蛋白之间的相互作用在运输中的重要性。有关转导素及其与视紫红质复合物的单分子行为的论文正在准备中。少

The main purpose of this research is to clarify the operating mechanism of G protein transducin in vertebrate photoreceptor disk membranes by single molecular imaging technique. The second purpose is to examine the molecular mechanism of G protein trafficking between inner and outer segment of rod photoreceptor during light- or dark adaptation. As the first task to clear, PI challenged to label transducin with near-infrared fluorescent compound (Hilyte 750), and succeeded. The labeled transducin retained its normal activity in binding light-bleached rhodopsin, nucleotide exchange activity, and PDE6 activation. By using this fluorescent-labeled transducin, Pl succeeded to observe single molecular motion of transducin in the dark state by using 750 nm laser beam for excitation of the label. Transducin showed much faser diffusion than rhodopsin in dark-adapted disk membrane. Upon exposure to visible light, transducin forms complex with light-bleached rhodopsin, and the complex showed slow … More er diffusion than rhodopsin. The diffusion rate of the complex was dependent on cholesterol-content of the disk membrane, i.e. the cholesterol-depletion by cholesterol-binding compound methyl-beta-cyclodextrin speeded up the diffusion of transducin-rhodopsin complex, suggesting that the activation of transducin occurs at the peripheries of cholesterol-dependent microdomains in the disk membrane. Although the mechanism of the microdomain formation has not been elucidated, these result gave us a deeper and new insight I into the G protein signaling mechanism of vertebrate phototransduction system. As for the trafficking of transducin between inner and outer segment of rod photoreceptor, Pl has not been able to introduce fluorescent-labeled transducin into rod outer segment. However, Pl's finding that GTP-form transducin shows propensity to form aggregation at the peripheral spots seemingly tubulin fibers at the mouth of incisures of the disk membrane suggested the importance of the interaction between transducin with tubulin in the trafficking. Papers about the single molecular behavior of transducin and its complex with rhodopsin are now in preparation. Less