CYSTEINE RESIDUE-110 AND RESIDUE-187 ARE ESSENTIAL FOR THE FORMATION OF CORRECT STRUCTURE IN BOVINE RHODOPSIN
CYSTEINE RESIDUE-110 AND RESIDUE-187 ARE ESSENTIAL FOR THE FORMATION OF CORRECT STRUCTURE IN BOVINE RHODOPSIN
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DOI：
10.1073/pnas.85.22.8459
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发表时间：
1988-11-01
影响因子：
11.1
通讯作者：
KHORANA, HG
中科院分区：
文献类型：
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作者：
KARNIK, SS;SAKMAR, TP;KHORANA, HG
为了研究不同半胱氨酸残基在牛视紫红质中的作用，制备了一系列突变体，其中半胱氨酸残基被系统地替换为丝氨酸。突变基因在猴肾细胞（COS-1）中表达，并评估突变视蛋白的表达水平、糖基化模式以及形成视紫红质发色团特征和激活转导蛋白的能力。三个细胞质半胱氨酸（Cys-316、Cys-322 和 Cys-323）和四个膜嵌入半胱氨酸（Cys-140、Cys-167、Cys-222 和 Cys-264）的取代产生了具有野生型表型的蛋白质。此外，Cys-185 的单一取代产生了野生型表型。相反，三个椎间盘内半胱氨酸（Cys-110、Cys-185 和 Cys-187）的取代或 Cys-110 或 Cys-187 的单一取代产生的蛋白质表达水平降低、糖基化异常且无法结合 11-顺式视网膜。因此，在牛视紫红质的 10 个半胱氨酸中，只有椎间盘内的 Cys-110 和 Cys-187 对于蛋白质的正确三级结构至关重要。
To investigate the role of different cysteine residues in bovine rhodopsin, a series of mutants were prepared in which the cysteine residues were systematically replaced by serines. The mutant genes were expressed in monkey kidney cells (COS-1) and the mutant opsins were evaluated for their levels of expression, glycosylation patterns, and ability to form the chromophore characteristic of rhodopsin and to activate transducin. Substitution of the three cytoplasmic cysteines (Cys-316, Cys-322, and Cys-323) and the four membrane-embedded cysteines (Cys-140, Cys-167, Cys-222, and Cys-264) produced proteins with wild-type phenotype. Also, single substitutions of Cys-185 gave rise to a wild-type phenotype. In contrast, substitution of the three intradiscal cysteines (Cys-110, Cys-185, and Cys-187) or single substitution of Cys-110 or Cys-187 gave proteins that were expressed at reduced levels, glycosylated abnormally, and unable to bind 11-cis-retinal. Thus, of the 10 cysteines in bovine rhodopsin, only intradiscal Cys-110 and Cys-187 are essential for the correct tertiary structure of the protein.