Morizumi Takefumi, Imai Hiroo, Shichida Yoshinori
Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan, and CREST, Japan Science and Technology Agency, Japan.
Biochemistry. 2005 Jul 26;44(29):9936-43. doi: 10.1021/bi0504512.
Rhodopsin is a photoreceptive protein that is present in rod photoreceptor cells, inducing a GDP-GTP exchange reaction on the retinal G-protein transducin (Gt) upon light absorption. This exchange reaction proceeds through at least three steps, which include the binding of photoactivated rhodopsin to GDP-bound Gt, the dissociation of GDP from the rhodopsin-Gt complex, and the binding of GTP to the nucleotide-unbound Gt. These steps have been thought to occur after the formation of the rhodopsin intermediate, meta-II; however, the extra formation of meta-II, which reflects the formation of a complex with Gt, was inhibited in the presence of excess GDP. Here, we use a newly developed CCD spectrophotometer to show that a meta-II precursor, meta-Ib, which has an absorption maximum at visible region, can bind to Gt in its GDP-bound form in urea-washed bovine rod outer segment membranes. The affinity of meta-Ib for GDP-bound Gt is about two times less than that of meta-II for GDP-unbound Gt, indicating that the extra formation of meta-II is observed at equilibrium even in the presence of the meta-Ib-Gt complex. This is the first identification of a complex that includes the GDP-bound form of G protein. Our results strongly suggest that the protein conformational change of the rhodopsin intermediate after binding to Gt is important for the induction of the nucleotide release from the alpha-subunit of Gt.
视紫红质是一种存在于视杆光感受器细胞中的光感受蛋白，在吸收光后会诱导视网膜G蛋白转导素（Gt）发生GDP-GTP交换反应。这种交换反应至少通过三个步骤进行，包括光激活的视紫红质与结合GDP的Gt结合、GDP从视紫红质-Gt复合物中解离以及GTP与未结合核苷酸的Gt结合。这些步骤被认为是在视紫红质中间体meta-II形成之后发生的；然而，在存在过量GDP的情况下，反映与Gt形成复合物的meta-II的额外形成受到抑制。在这里，我们使用新开发的电荷耦合器件（CCD）分光光度计表明，在可见区域具有最大吸收峰的meta-II前体meta-Ib可以在尿素洗涤的牛视杆外段膜中以其结合GDP的形式与Gt结合。meta-Ib对结合GDP的Gt的亲和力比对未结合GDP的Gt的meta-II的亲和力低约两倍，这表明即使在存在meta-Ib-Gt复合物的情况下，在平衡时也能观察到meta-II的额外形成。这是首次鉴定出包含结合GDP形式的G蛋白的复合物。我们的结果强烈表明，视紫红质中间体与Gt结合后的蛋白质构象变化对于诱导Gt的α亚基释放核苷酸很重要。
