Isolation and functional characterization of a stable complex between photoactivated rhodopsin and the G protein, transducin
Isolation and functional characterization of a stable complex between photoactivated rhodopsin and the G protein, transducin
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DOI：
10.1096/fj.07-114835
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发表时间：
2009-02-01
期刊：
影响因子：
4.8
通讯作者：
Palczewski, Krzysztof
中科院分区：
文献类型：
--
作者：
Jastrzebska, Beata;Golczak, Marcin;Palczewski, Krzysztof
光活化视紫红质（Rho * 或Meta II）和G蛋白转导素（Gt-GDP）之间的瞬时结合是视觉信号级联中的第一步。光引起视紫红质（Rho）中的11-顺式-亚视黄基发色团光异构化为全反式-亚视黄基，这诱导构象变化，使得Gt-GDP对接到Rho * 表面上。然后GDP从Gt解离，在GTP结合之前留下瞬时核苷酸空的Rho *-Gt（e）复合物，然后Gt-GTP从Rho * 解离。在各种Rho *-GT复合物的结构研究开始之前，需要进一步的生物化学进展。在这里，我们描述了通过蔗糖梯度离心分离正十二烷基-β-麦芽糖苷溶解的、稳定的、功能活性的Rho *-Gt（e）、Rhoe *-Gt（e）和9-顺式-视黄醛/11-顺式-视黄醛再生Rho-Gt（e）复合物。在这些复合物中，Rho * 在光谱上保持在其Meta II状态，并且Gt（e）保留其与GTP γ S相互作用的能力。从Rho *-Gt（e）中去除全反式-视黄叉基对Rho（e）*-Gt（e）复合物的稳定性没有影响。此外，具有Gt中的空核苷酸结合口袋和Rho中的空类视色素结合口袋的Rho（e）*-Gt（e）复合物中的视蛋白再生高达75%而没有复合物解离。这些结果表明，一旦Rho * 与Gt偶联，发色团在稳定该复合物中起次要作用。此外，在用9-顺式-视黄醛/11-顺式-视黄醛再生的复合物中，Rho保留了类似于由Gte apo蛋白稳定的Rho * 的构象。- Jastrzebska，B.，Golczak，M.，Fotiadis，D.，Engel，A.，和Palczewski，K..光活化视紫红质与G蛋白转导素之间稳定复合物的分离和功能表征。FASEB J. 23，371 - 381（2009）
Transitory binding between photoactivated rhodopsin (Rho* or Meta II) and the G protein transducin (Gt-GDP) is the first step in the visual signaling cascade. Light causes photoisomerization of the 11-cis-retinylidene chromophore in rhodopsin (Rho) to all-trans-retinylidene, which induces conformational changes that allow Gt-GDP to dock onto the Rho* surface. GDP then dissociates from Gt, leaving a transient nucleotide-empty Rho*-Gt(e) complex before GTP becomes bound, and Gt-GTP then dissociates from Rho*. Further biochemical advances are required before structural studies of the various Rho*-Gt complexes can be initiated. Here, we describe the isolation of n-dodecyl-beta-maltoside solubilized, stable, functionally active, Rho*-Gt(e), Rhoe*-Gt(e), and 9-cis-retinal/11-cis-retinal regenerated Rho-Gt(e) complexes by sucrose gradient centrifugation. In these complexes, Rho* spectrally remained in its Meta II state, and Gt(e) retained its ability to interact with GTP gamma S. Removal of all-trans-retinylidene from Rho*-Gt(e) had no effect on the stability of the Rho(e)*-Gt(e) complex. Moreover, opsin in the Rho(e)*-Gt(e) complex with an empty nucleotide-binding pocket in Gt and an empty retinoid-binding pocket in Rho was regenerated up to 75% without complex dissociation. These results indicate that once Rho* couples with Gt, the chromophore plays a minor role in stabilizing this complex. Moreover, in complexes regenerated with 9-cis-retinal/11-cis-retinal, Rho retains a conformation similar to Rho* that is stabilized by Gte apo-protein. - Jastrzebska, B., Golczak, M., Fotiadis, D., Engel, A., and Palczewski, K.. Isolation and functional characterization of a stable complex between photoactivated rhodopsin and the G protein, transducin. FASEB J. 23, 371-381 (2009)