Structure and function in rhodopsin: replacement by alanine of cysteine residues 110 and 187, components of a conserved disulfide bond in rhodopsin, affects the light-activated metarhodopsin II state.
A disulfide bond that is evidently conserved in the guanine nucleotide-binding protein-coupled receptors is present in rhodopsin between Cys-110 and Cys-187. We have replaced these two cysteine residues by alanine residues and now report on the properties of the resulting rhodopsin mutants. The mutant protein C110A/C187A expressed in COS cells resembles wild-type rhodopsin in the ground state. It folds correctly to bind 11-cis-retinal and form the characteristic rhodopsin chromophore. It is inert to hydroxylamine in the dark, and its stability to dark thermal decay is reduced, relative to that of the wild type, by a delta delta G not equal to of only -2.9 kcal/mol. Further, the affinities of the mutant and wild-type rhodopsins to the antirhodopsin antibody rho4D2 are similar, both in the dark and in light. However, the metarhodopsin II (MII) and MIII photointermediates of the mutant are less stable than those formed by the wild-type rhodopsin. Although the initial rates of transducin activation are the same for both mutant and wild-type MII intermediates at 4 degrees C, at 15 degrees C the MII photointermediate in the mutant decays more than 20 times faster than in wild type. We conclude that the disulfide bond between Cys-110 and Cys-187 is a key component in determining the stability of the MII structure and its coupling to transducin activation.
Davidson
F F
FF
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Loewen
P C
PC
Khorana
H G
HG
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides; D006150:Guanine Nucleotides / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D011994:Recombinant Proteins; D012243:Rhodopsin / Q000737:chemistry* / Q000276:immunology / Q000502:physiology*; D017299:Rod Opsins / Q000737:chemistry; D013329:Structure-Activity Relationship; D013696:Temperature; D015293:Transducin / Q000378:metabolism
D004220:Disulfides; D006150:Guanine Nucleotides; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D017299:Rod Opsins; D012243:Rhodopsin; D015293:Transducin; D003545:Cysteine; D000409:Alanine