SEQUENCE VARIABILITY IN THE RETINAL-ATTACHMENT DOMAIN OF MAMMALIAN RHODOPSINS
SEQUENCE VARIABILITY IN THE RETINAL-ATTACHMENT DOMAIN OF MAMMALIAN RHODOPSINS
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DOI：
10.1042/bj2170605
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发表时间：
1984-01-01
影响因子：
4.1
通讯作者：
FINDLAY, JBC
中科院分区：
文献类型：
--
作者：
PAPPIN, DJC;FINDLAY, JBC
用11-cis-[15- 3 H]retinal再生绵羊视紫红质，并用金黄色葡萄球菌V8蛋白酶原位切割，得到Mr [分子比] 27000（V8-L）和12000（V8-S）的2个膜结合片段。用伴刀豆球蛋白A-Sepharose 4 B亲和层析纯化蛋白水解复合物后，用硼氢化物还原[3 H]retinal与蛋白共价连接。用CNBr和三氟乙酸裂解纯化的[3 H]-视黄基V8-S片段，通过凝胶过滤分离所得肽，并对[3 H]视黄基肽进行测序。为分离和测序绵羊蛋白质的该区域而开发的方案直接且可重复地应用于漂白和未再生的猪和马视蛋白。片段的一级结构的比较揭示了紧接在羊蛋白中显示的赖氨酸残基之后的序列中的显著变化，该赖氨酸残基是发色团的醛基的附着点。突变位置位于先前预测为采用不规则或扭曲构象的区域中，并暗示可能提供对视觉色素的光谱和功能特性的更好理解的结构排列。
Ovine rhodopsin was regenerated with 11-cis-[15-3H]retinal and cleaved in situ by Staphylococcus aureus V8 proteinase to give 2 membrane-bound fragments of Mr [molecular ratio] 27000 (V8-L) and 12000 (V8-S). After purification of the proteolysed complex by affinity chromatography with concanavalin A-Sepharose 4B, [3H]retinal was covalently linked to the protein by reduction with borohydride. The purified [3H]-retinyl V8-S fragment was cleaved with CNBr and trifluoroacetic acid, the resulting peptides resolved by gel filtration and the [3H]retinyl peptide sequenced. The protocol developed for the isolation and sequencing of this region of the ovine protein was applied directly, and reproducibly, to bleached and unregenerated porcine and equine opsins. Comparisons of the primary structures of the fragments reveals marked variation in the sequence immediately after the lysine residue shown in the ovine protein to be the attachment point for the aldehyde group of the chromophore. Mutable positions are localized in regions previously predicted as adopting nonregular or distorted conformations and hint at structural arrangements that may provide a better understanding of the spectral and functional properties of the visual pigment.