Formation of the meta II photointermediate is accompanied by conformational changes in the cytoplasmic surface of rhodopsin.
Formation of the meta II photointermediate is accompanied by conformational changes in the cytoplasmic surface of rhodopsin.
复制标题
meta II 光中间体的形成伴随着视紫红质细胞质表面的构象变化。
DOI：
10.1021/bi00096a012
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Khorana,HG
中科院分区：
文献类型：
--
作者：
Resek,JF;Farahbakhsh,ZT;Hubbell,WL;Khorana,HG
摘要：已经产生了视紫红质的五种突变，每种突变在胞质结构域的62、65、140、240或316位含有独特的半胱氨酸残基。单一的反应性半胱氨酸衍生巯基特定的氮氧自由基自旋标记，和电子顺磁共振（EPR）光谱进行了分析，在黑暗中和光漂白后，在月桂基麦芽糖苷和毛地黄皂苷。极性和非极性顺磁剂的碰撞率表明，他们都暴露在水环境中。光漂白的突变体毛地黄皂苷，其中逮捕的蛋白质在Meta我中间，产生的流动性的连接氮氧化物的变化不大。另一方面，在月桂基麦芽糖苷的光漂白产生的Meta II中间体和显着变化的EPR光谱的氮氧化物连接到位置140和316。视紫红质是脊椎动物视杆细胞中的膜结合光感受器，含有1条348个氨基酸的多肽链，其序列已通过cDNA和蛋白质测序确定（Ovchinnikov等，1982; Hargrave等人，1983; Nathans & Hogness，1983）。二级结构模型，假设蛋白质含有7个跨膜螺旋，如图1所示。发色团11-cw-retinal位于膜包埋的螺旋簇中，通过质子化的Schiff碱与Lys 296 inhelix G连接。螺旋C中的谷氨酸113充当亚视黄基席夫碱的抗衡物，使螺旋C和G彼此并置（Sakmar等人，1989; Zhokovsky & Oprian，1989; Nathans，1990）。细胞质螺旋间环（C-*-D、E-* F和G-* 羧基末端）含有转导素的光依赖性结合位点（Konig等人，1989 a; Franke等人，1990，1992）（Gt）1，而羧基尾含有光依赖性磷酸化的位点（Kuhn，
Revised Manuscript Received August 31, 1993® abstract: Five mutations of rhodopsin have been produced, each of which contains a unique cysteine residue at positions 62, 65,140, 240, or 316 in the cytoplasmic domain. The single reactive cysteines were derivatized with a sulfhydryl-specific nitroxide spin-label, and the electron paramagnetic resonance (EPR) spectra were analyzed in both lauryl maltoside and digitonin in the dark and after photobleaching. The collision rate of the attached nitroxides with polar and nonpolar paramagnetic agents indicated that they were all exposed to the aqueous environment. Photobleaching of the mutants in digitonin, which arrests the protein at the meta I intermediate, produced little change in mobility of the attached nitroxide. On the other hand, photobleaching in lauryl maltoside produced the meta II intermediate and significant changes in the EPR spectra of the nitroxides attached to positions 140 and 316. These data directly reveal a light-induced conformational change in the cytoplasmic loops that accompanies meta II formation.Rhodopsin, the membrane-bound photoreceptor in the vertebrate rod cell, contains 1 polypeptide chain of 348 amino acids whose sequence has been determined by cDNA and protein sequencing (Ovchinnikov et al., 1982; Hargrave et al., 1983; Nathans & Hogness, 1983). A secondary structure model, assuming that the protein contains seven transmembrane helices, is shown in Figure 1. The chromophore 11-cw-retinal, located in the membrane-embedded helical cluster, is linked to Lys296 inhelix G via a protonated Schiff base. Glutamic acid 113, in helix C, serves as the counterion to the retinylidene Schiff base placing helix C and G in apposition to each other (Sakmar et al., 1989; Zhokovsky & Oprian, 1989; Nathans, 1990). The cytoplasmic interhelical loops (C-*-D, E—* F, and G-* carboxyl terminus) contain the light-dependent binding site for transducin (Konig et al., 1989a; Franke et al., 1990, 1992)(Gt) 1 whereas the carboxyl tail contains the sites for light-dependent phosphorylation (Kuhn,