Environment around the chromophore in pharaonis phoborhodopsin: mutation analysis of the retinal binding site.
Phoborhodopsin (pR or sensory rhodopsin II, sRII) and pharaonis phoborhodopsin (ppR or pharaonis sRII, psRII) have a unique absorption maximum (lambda(max)) compared with three other archaeal rhodopsins: lambda(max) of pR and ppR is approx. 500 nm and of others (e.g. bacteriorhodopsin, bR) is 560-590 nm. To determine the residue contributing to the opsin shift from ppR to bR, we constructed various ppR mutants, in which a single residue was substituted for a residue corresponding to that of bR. The residues mutated were those which differ from that of bR and locate within 5 A from the conjugated polyene chain of the chromophore or any methyl group of the polyene chain. The shifts of lambda(max) of all mutants were small, however. We constructed a mutant in which all residues which differ from those of bR in the retinal binding site were simultaneously substituted for those of bR, but the shift was only from 499 to 509 nm. Next, we constructed a mutant in which 10 residues located within 5 A from the polyene as described above were simultaneously substituted. Only 44% of the opsin shift (lambda(max) of 524 nm) from ppR to bR was obtained even when all amino acids around the chromophore were replaced by the same residues as bR. We therefore conclude that the structural factor is more important in accounting for the difference of lambda(max) between ppR and bR rather than amino acid substitutions. The possible structural factors are discussed.
Shimono
K
K
Laboratory of Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, 060-0812, Sapporo, Japan.
Ikeura
Y
Y
Sudo
Y
Y
Iwamoto
M
M
Kamo
N
N
D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D002338:Carotenoids / Q000737:chemistry* / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D004252:DNA Mutational Analysis; D025602:Halorhodopsins; D008958:Models, Molecular; D009154:Mutation; D012172:Retinaldehyde / Q000737:chemistry*; D025603:Sensory Rhodopsins; D013053:Spectrophotometry
D019843:Archaeal Proteins; D025602:Halorhodopsins; D025603:Sensory Rhodopsins; C061508:sensory rhodopsin II protein, archaeal; D002338:Carotenoids; D012172:Retinaldehyde