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首页> 外文期刊>Biochemistry >Rhodopsin-interacting surface of the transducin gamma subunit
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Rhodopsin-interacting surface of the transducin gamma subunit

机译：转导蛋白γ亚基的视紫红质相互作用表面

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The visual signaling pathway is initiated by photoactivation of the GPCR rhodopsin, which activates nucleotide exchange on the heterotrimeric G-protein transducin (Gt). Domains on both Gt alpha and Gt beta gamma subunits participate in coupling to rhodopsin. Previously, we have shown by high-resolution NMR that the farnesylated C-terminal peptide of G gamma (60-71), DKNPFKELKGGC, assumes an amphipathic helical conformation during interaction with metarhodopsin II [Kisselev, O. G., and Downs, M. A. (2003) Structure 11, 367-373]. This conformation was docked to the structure of holo-Gt to create a model of rhodopsin-Gt interaction. Here we test this model by mutational analysis of Gt. To evaluate the contribution of specific amino acids of the Gt gamma C-terminal region involved in binding and GTP-dependent release of transducin from native rhodopsin membranes, we have systematically substituted each of the amino acids in the C-terminal region of Gt gamma for alanine. The mutants were co-expressed with six-histidine-tagged Gt beta subunits in Sf9 insect cells. The Gt beta-6-His-gamma mutant proteins were purified and assayed in the presence of Gt alpha for the GTP-dependent interactions with light-activated rhodopsin. Several of the alanine mutants, N62A, P63A, and F64A, exhibited significant functional defects at the level of R*-Gt complex formation. These data show that the conserved N-terminal end of the helical domain in the Gt gamma (60-71) region has the most significant effect on rhodopsin-Gt interactions, which places important constraints on the model of the rhodopsin-Gt complex.
机译：视觉信号转导途径是由GPCR视紫红质的光激活引发的，该视紫红质激活异三聚体G蛋白转导蛋白（Gt）上的核苷酸交换。 Gt alpha和Gt betaγ亚基上的结构域均参与视紫红质的偶联。以前，我们已通过高分辨率NMR显示，Gγ（60-71）的法尼基化C末端肽DKNPFKELKGGC在与变紫红质II（Kisselev，OG，and Downs，MA（2003））相互作用期间呈现两亲性螺旋构象。结构11，367-373]。将该构象对接至holo-Gt的结构，以创建视紫红质-Gt相互作用的模型。在这里，我们通过对Gt进行突变分析来测试该模型。为了评估参与结合和GTP依赖性释放天然视紫红质膜的GtγC末端区域的特定氨基酸的贡献，我们已系统地替换了GtγC末端区域的每个氨基酸丙氨酸。突变体与六个组氨酸标记的Gtβ亚基在Sf9昆虫细胞中共表达。纯化Gt beta-6-His-gamma突变蛋白，并在Gt alpha存在下分析与光活化视紫红质的GTP依赖性相互作用。几种丙氨酸突变体N62A，P63A和F64A在R * -Gt复合物形成的水平上表现出明显的功能缺陷。这些数据表明，Gtγ（60-71）区域中螺旋结构域的保守N末端对视紫红质-Gt相互作用具有最重要的影响，这对视紫红质-Gt复合体的模型施加了重要限制。

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