Mapping of the amino acids in the cytoplasmic loop connecting helices C and D in rhodopsin. Chemical reactivity in the dark state following single cysteine replacements.
Mapping of the amino acids in the cytoplasmic loop connecting helices C and D in rhodopsin. Chemical reactivity in the dark state following single cysteine replacements.
复制标题
视紫红质中连接螺旋 C 和 D 的细胞质环中的氨基酸图谱。
DOI：
10.1021/bi00027a032
复制
发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Khorana,HG
中科院分区：
文献类型：
--
作者：
Ridge,KD;Zhang,C;Khorana,HG
Revised Manuscript Received April 17, 1995® abstract: The cytoplasmic loop connecting helices C and Din rhodopsin is a part of the region involved in protein—protein interactions during signal transduction. To probe the structure of the CD loop, we have replaced, one at a time, the amino acids136—150 by cysteineresidues. The cysteine substitution mutants contained only the introduced single reactive cysteines and were prepared from a base opsin mutant that retained only the three intradiscal cysteines. Allof the cysteine substitution mutants formed the characteristic rhodopsin chromophore (Zmax, 500 nm) with 11-cis-retinal. They showed normal photobleaching characteristics and activated transducin in a light-dependent manner, albeit at lower levels than the wild-type pigment. The newly introduced cysteines in the substitution mutants all underwent alkylation in the dark with the membrane-permeant sulfhydryl reagent A-ethylmaleimide, but with varying rates. The cysteine substitution mutants also showed prominent differences in alkylation with membrane-impermeant A-polymethylenecarboxylmaleimides of various alkyl chain lengths. Notably, derivatization of the cysteines in the mutants was not observed with the polar sulfhydryl reagents iodoacetic acid or iodoacetamide. These findingshighlight intrinsic differences in both the reactivity and accessibility of the different cysteine residues in the CD loop and support the important role for a structure in the second cytoplasmic region of rhodopsin.Rhodopsin, the photoreceptor of the vertebraterod cell, is assumed to undergo a conformational change upon light activation. This allows the bindingof transducin (Gt) 1 on the cytoplasmic face, the first step in the ensuing biochemical cascade (Kuhn & Hargrave, 1981; Bennet et al., 1982). The latter culminates in the closing of the cation conductance channels in the plasma membrane, causing hyperpolarization of the rod cell (Fesenko et al., 1985). The nature of the structural changes in rhodopsin occurring upon light activa-tion are essentially unknown. Therefore, we are interested in developing approaches to the study of these presumed structural changes. One potentially useful approach is to introduce single cysteine residues by replacement of the naturally occurring amino acids at predetermined positions in the different domains of rhodopsin. The sulfhydryl groups in the cysteine residues then provide a handle for chemical and biophysical investigations. Previous studies along this line with bacteriorhodopsin proved to be very rewarding (Flitsch & Khorana, 1989; Altenbach et al., 1989, 1990; Greenhalgh et al., 1991; Steinhoff et al., 1994). The importance of the cytoplasmic loops (Figure 1) for the rhodopsin—Gj interaction has been highlighted by a
