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首页> 外文期刊>Biochemistry >Direct observation of the complex formation of GDP-bound transducin with the rhodopsin intermediate having a visible absorption maximum in rod outer segment membranes
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Direct observation of the complex formation of GDP-bound transducin with the rhodopsin intermediate having a visible absorption maximum in rod outer segment membranes

机译：直接观察GDP结合的转导蛋白与视紫红质中间体在杆外节膜中具有最大可见吸收的复合物形成

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摘要

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.
机译：视紫红质是一种存在于杆状感光细胞中的感光蛋白，在光吸收后可诱导视网膜G蛋白转导蛋白（Gt）发生GDP-GTP交换反应。该交换反应至少通过三个步骤进行，包括光活化的视紫红质与GDP结合的Gt的结合，GDP与视紫红质-Gt复合物的解离以及GTP与核苷酸未结合的Gt的结合。人们认为这些步骤是在视紫红质中间体meta-II形成之后发生的。然而，在过量GDP的存在下，额外的meta-II的形成被抑制了，这反映了与Gt形成复合物的形成。在这里，我们使用新开发的CCD分光光度计显示，在可见光区具有最大吸收的meta-II前体meta-Ib可以与GDP结合的Gt在尿素清洗的牛杆外段膜中结合。 。 meta-Ib对与GDP结合的Gt的亲和力约比meta-II对与GDP结合的Gt的亲和力小两倍，这表明即使在存在meta-Ib的情况下，也可以在平衡状态下观察到meta-II的额外形成。 -Gt复合体。这是对复合物的首次鉴定，其中包括与GDP结合的G蛋白形式。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从Gt的α亚基释放核苷酸非常重要。

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