视紫红质中的单个氨基酸替换（赖氨酸248变为亮氨酸）会阻止转导蛋白的激活。

A single amino acid substitution in rhodopsin (lysine 248----leucine) prevents activation of transducin.

作者信息

Franke R R, Sakmar T P, Oprian D D, Khorana H G

机构信息

Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.

出版信息

J Biol Chem. 1988 Feb 15;263(5):2119-22.

PMID:3123487

Abstract

In structure-function studies on bovine rhodopsin by in vitro site-specific mutagenesis, we have prepared three mutants in the cytoplasmic loop between the putative transmembrane helices E and F. In each mutant, charged amino acid residues were replaced by neutral residues: mutant 1, Glu239----Gln; mutant 2, Lys248----Leu; and mutant 3, Glu247----Gln, Lys248----Leu, and Glu249----Gln. The mutant rhodopsin genes were expressed in monkey kidney (COS-1) cells. After the addition of 11-cis-retinal to the cells, the rhodopsin mutants were purified by immunoaffinity adsorption. Each mutant gave a wild-type rhodopsin visible absorption spectrum. The mutants were assayed for their ability to stimulate the GTPase activity of transducin in a light-dependent manner. While mutants 1 and 3 showed wild-type activity, mutant 2 (Lys248----Leu) was inactive.

摘要

在通过体外定点诱变对牛视紫红质进行的结构-功能研究中，我们在假定的跨膜螺旋E和F之间的胞质环中制备了三个突变体。在每个突变体中，带电荷的氨基酸残基被中性残基取代：突变体1，Glu239→Gln；突变体2，Lys248→Leu；突变体3，Glu247→Gln、Lys248→Leu和Glu249→Gln。突变的视紫红质基因在猴肾（COS-1）细胞中表达。向细胞中加入11-顺式视黄醛后，通过免疫亲和吸附纯化视紫红质突变体。每个突变体都给出了野生型视紫红质的可见吸收光谱。检测了这些突变体以光依赖方式刺激转导素GTP酶活性的能力。虽然突变体1和3表现出野生型活性，但突变体2（Lys248→Leu）无活性。