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

本研究的主要目的是通过单分子成像技术阐明G蛋白转导蛋白在脊椎动物光感受器盘膜中的作用机制。第二个目的是研究杆状光感受器内外段在光或暗适应过程中G蛋白运输的分子机制。作为第一个明确的任务，PI挑战用近红外荧光化合物（Hilyte 750）标记转导蛋白，并取得了成功。标记的转导蛋白在结合光漂紫质、核苷酸交换活性和PDE6活化方面保持正常活性。利用这种荧光标记的转导蛋白，Pl利用750 nm激光束激发标记，成功地观察了转导蛋白在暗态下的单分子运动。转导蛋白在暗适应盘膜上的扩散速度明显快于视紫红质。在可见光下，转导蛋白与光漂白的视紫红质形成复合物，复合物比视紫红质扩散缓慢。复合物的扩散速率依赖于盘膜中胆固醇含量，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-视紫红质复合物的扩散，这表明转导蛋白的激活发生在盘膜中胆固醇依赖微域的外围。虽然微结构域形成的机制尚未阐明，但这些结果使我们对脊椎动物光转导系统的G蛋白信号传导机制有了更深入、新的认识。对于杆状光感受器内外段之间的转导素的运输，Pl还不能将荧光标记的转导素引入杆状光感受器外段。然而，Pl发现gtp形式的转导蛋白倾向于在外周点形成聚集，似乎是在磁盘膜的开口处的微管蛋白纤维，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。关于转导蛋白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