Rhodopsin-G-protein interactions monitored by resonance energy transfer.
Rhodopsin-G-protein interactions monitored by resonance energy transfer.
复制标题
通过共振能量转移监测视紫红质-G-蛋白相互作用。
DOI：
10.1021/bi00430a043
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发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Montal,M
中科院分区：
文献类型：
--
作者：
Borochov-Neori,H;Montal,M
加州大学生物学和物理学系，圣地亚哥，拉霍亚，加州92093接收1988年4月4日;修订的Mandarin pt接收1988年10月20日摘要：实施共振能量转移测量以监测用纯化蛋白质重构的磷脂囊泡中G蛋白和视紫红质之间的特异性相互作用。从与几种巯基试剂反应的漂白棒外节（ROS）中提取荧光标记的G蛋白：7V-（1-芘基）马来酰亚胺（P）、monobromobimane（B）、7-（二乙基氨基）-3-（4-马来酰亚胺基苯基）-4-甲基香豆素（C）和7V-（4-苯胺基-1-萘基）马来酰亚胺（A）。ROS的有限标记，导致每个G蛋白分子的修饰少于一个SH残基，每个视紫红质的修饰少于0.2个残基，并没有损害视紫红质和G蛋白之间的特异性原位相互作用。这是通过保存它们的光激活紧密结合和Gpp（NH）p结合以及它们与过量GTP的快速解离来证明的。荧光标记物在G蛋白3个亚基中的分布表明，当G蛋白与漂白视紫红质特异性结合时，在7个亚基中有一个高活性的SH基团可被标记。纯化的G-蛋白的荧光衍生物与纯化的视紫红质在脂质囊泡中重组的纯化的还原恢复了光激活的Gpp（NH）p结合到与未标记的G-蛋白测量的水平相当。在ROS耗尽外周蛋白的情况下获得了类似的观察结果。同样，多达两个SH基团的修饰每个视紫红质分子的荧光试剂不影响功能重组的G-蛋白与视紫红质在重建的脂质囊泡或在耗尽的ROS。视紫红质和G蛋白之间的相互作用通过共振能量转移测量来监测，以下荧光缀合物作为供体/受体对：P-视紫红质/CG-蛋白，P-视紫红质/BG-蛋白和PG-蛋白/C-视紫红质。在这些对的漂白重组体中检测到能量转移，反映了G蛋白与膜的结合。G蛋白的特异性结合和非特异性结合通过过量的（a）GTP的不可水解类似物（其促进视紫红质-G蛋白复合物的解离）和（B）未标记的G蛋白（其竞争视紫红质中的结合位点）诱导的能量转移的减少来辨别。在第一近似值中，这些测量将G蛋白和视紫红质38-46 A的标记-SH基团分开。视紫红质对视网膜视杆细胞内环GMP磷酸二酯酶的快速和高度放大激活是视觉信号转导的一个中心过程。随后的环GMP水解导致光感受器质膜中Na+通道的关闭[Fesenko et al.，1985; Yau和Nakatani，1985; Haynes等人，1986年;齐默尔曼和贝勒，1986年;审查斯特雷尔（1986年）]。磷酸二酯酶的光活化需要GTP结合蛋白（G蛋白）的存在。1 G蛋白的偶联作用是通过视紫红质和G蛋白的构象和相互作用的一系列变化来实现的。G蛋白以高亲和力结合到光解的视紫红质（Godchaux &齐默尔曼，1979; Kuhn，1980），经历构象变化（Fung & Nash，1983; Halliday等人，1984; Navon & Fung，1984），随后交易所将GDP与GTP绑定（Godchaux &齐默尔曼，1979; Kuhn，1980; Fung & Stryer，1980）。激活的G蛋白-GTP复合物从激发的视紫红质中解离（Kuhn，1980，1984）并激活...
Departments of Biology and Physics, University of California, San Diego, LaJolla, California 92093 Received April 4, 1988; Revised Manuscript Received October 20, 1988 abstract: Resonance energy transfer measurements were implemented to monitor the specific interactions between G-protein and rhodopsin in phospholipid vesicles reconstituted with the purified proteins. Fluorescently labeled G-protein was extracted from bleached rod outer segments (ROS) reacted with several sulfhydryl reagents: 7V-(1-pyrenyl) maleimide (P), monobromobimane (B), 7-(diethylamino)-3-(4-maleimidylphenyl)-4-methylcoumarin (C), and 7V-(4-anilino-l-naphthyl) maleimide (A). Limited labeling of ROS, resulting in the modification of less than a single-SH residue per G-protein moleculeand less than 0.2 residue per rhodopsin, did not impair thespecific in situ interactions between rhodopsin and G-protein. This was demonstrated by preservation of their light-activated tight association and Gpp (NH) p binding and their fast dissociation with excess GTP. The distribution of fluorescent label among the three subunits of G-protein revealed a highly reactive-SH group in the 7 subunit accessible to labeling when G-protein was bound specifically to bleached rhodopsin. Recombination of purifiedfluorescent derivatives of G-protein with purified rhodopsin reconstituted in lipid vesicles restoredthe light-activated Gpp (NH) p binding to a level comparable to that measured with unlabeled G-protein. Similar observations were obtained with ROS depleted of peripheral proteins. Likewise, modification of upto two-SH groups per rhodopsin molecule with the fluorescent reagents did not affect the functional recombination of G-protein with rhodopsin in reconstituted lipid vesicles or in depleted ROS. Interactions between rhodopsin and G-protein were monitored by resonance energy transfer measurements, with the following fluorescent conjugates as donor/acceptor couples: P-rhodopsin/CG-protein, P-rhodopsin/BG-protein, and PG-protein/C-rhodopsin. Energy transfer was detected in bleached recombinants of these pairs, reflecting the binding of G-protein to membranes. Specific binding and nonspecific binding of G-protein were discerned by the reduction of energy transfer induced by an excess of (a) nonhydrolyzable analogues of GTP, which promote the dissociation of the rhodopsin-G-protein complex, and (b) unlabeled G-protein, which competes for binding sites in rhodopsin. To a first approximation, these measurements placed the labeled-SH groups of G-protein and rhodopsin 38-46 A apart. e fast and highly amplified activation of the cyclic-GMP phosphodiesterase inretinal rodcells by excited rhodopsin is a central process in visual transduction. The subsequent hy-drolysis of cyclic GMP leads tothe closure of the Na+ channels in the photoreceptor plasma membrane [Fesenko et al., 1985; Yau and Nakatani, 1985; Haynes et al., 1986; Zimmerman & Baylor, 1986; reviewed by Stryer (1986)]. The photoactivation of the phosphodiesterase requires the presence of a GTP binding protein (G-protein). 1 The coupling role of the G-protein is achieved through a sequence of changes in the conformations and interactions of rhodopsin and G-protein. G-Protein binds with high affinity to photolyzed rhodopsin (Godchaux & Zimmerman, 1979; Kuhn, 1980), undergoes a conformational change (Fung & Nash, 1983; Halliday et al., 1984; Navon & Fung, 1984), and subsequently exchanges bound GDP for GTP (Godchaux & Zimmerman, 1979; Kuhn, 1980; Fung & Stryer, 1980). The activated G-protein-GTP complex dissociates from excited rhodopsin (Kuhn, 1980, 1984) and activates the …