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.
The cytoplasmic loop connecting helices C and D in 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 acids 136-150 by cysteine residues. 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. All of the cysteine substitution mutants formed the characteristic rhodopsin chromophore (lambda max, 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 N-ethylmaleimide, but with varying rates. The cysteine substitution mutants also showed prominent differences in alkylation with membrane-impermeant N-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 findings highlight 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.
Ridge
K D
KD
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.
Zhang
C
C
Khorana
H G
HG
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003545:Cysteine / Q000737:chemistry*; D003593:Cytoplasm / Q000737:chemistry*; D003624:Darkness; D005033:Ethylmaleimide / Q000737:chemistry; D019204:GTP-Binding Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D017433:Protein Structure, Secondary; D012243:Rhodopsin / Q000737:chemistry*
D012243:Rhodopsin; D019204:GTP-Binding Proteins; D003545:Cysteine; D005033:Ethylmaleimide