Assembly of functional rhodopsin requires a disulfide bond between cysteine residues 110 and 187.
Assembly of functional rhodopsin requires a disulfide bond between cysteine residues 110 and 187.
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DOI：
10.1016/s0021-9258(18)38195-x
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发表时间：
1990-10
期刊：
影响因子：
--
通讯作者：
S. Karnik;H. Khorana
中科院分区：
文献类型：
--
作者：
S. Karnik;H. Khorana
半胱氨酸残基110和187对于形成正确的牛视紫红质结构至关重要（Karnik, s.s., Sakmar, t.p., Chen， H.-B.）。，和Khorana， H. G. (1988) Proc. Natl。学会科学。《美国法典》第85卷，8459-8463页)。我们现在表明，这两个半胱氨酸残基的巯基相互作用形成一个二硫键。含半胱氨酸-丝氨酸取代的视紫红质突变体制备方法如下。在一个突变体CysVII中，视紫红质的10个半胱氨酸残基全部被丝氨酸取代。第二个突变体CysVIII只含有C110和C185；第三个突变体CysIX只含有C185和C187，而第四个突变体CysX只含有C110和C187。只有突变体cyx形成了功能性视紫红质。突变体CysVIII和CysIX与[3H]碘乙酸反应，显示出游离巯基的存在，而突变体CysX对该试剂无效。cyx与氰化物离子反应形成硫氰酸酯衍生物，显示出二硫键的存在。C110-C187二硫键被埋在紫红质中，因为与二硫还原剂和氰化物离子的反应需要事先用变性剂处理。
Cysteine residues 110 and 187 are essential for the formation of the correct bovine rhodopsin structure (Karnik, S. S., Sakmar, T. P., Chen, H.-B., and Khorana, H. G. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8459-8463). We now show that the sulfhydryl groups of these 2 cysteine residues interact to form a disulfide bond. Rhodopsin mutants containing cysteine—-serine substitutions were prepared as follows. In one mutant, CysVII, all the 10 cysteine residues of rhodopsin were replaced by serines. A second mutant, CysVIII, contained only C110 and C185; a third mutant, CysIX, contained only C185 and C187 while the fourth mutant, CysX, contained only C110 and C187. Only mutant CysX formed functional rhodopsin. Mutants CysVIII and CysIX reacted with [3H]iodoacetic acid showing the presence of free sulfhydryl groups while mutant CysX was inert to this reagent. CysX reacted with cyanide ion to form a thiocyanate derivative showing the presence of a disulfide bond. The C110-C187 disulfide bond is buried in rhodopsin because reactions with disulfide reducing agents and cyanide ion require prior treatment with denaturants.