V108M mutant of pharaonis phoborhodopsin: substitution caused no absorption change but affected its M-state.
Crystallographic data reveal that Met-118 in bacteriorhodopsin (bR) contacts directly with the C9 methyl group of retinal, and Khorana et al. [J. Biol. Chem. 268, 20305-20311 (1993)] suggest that this contact may regulate the absorption maximum (lambdamax). We have replaced the amino acid (Val-108) corresponding to Met-118 of bR by methionine in pharaonis phoborhodopsin (ppR), whose lambdamax is ca. 500 nm, while those of other bacterial rhodopsins such as bR, halorhodopsin, and sensory rhodopsin are red-shifted by 60-90 nm. By flash-photolysis measurement, we could not recognize a large spectral red-shift of the V108M mutant. On the other hand, the decay of ppRM (M-intermediate) of the mutant was approximately three times as fast as that of wild-type, and an M-like intermediate (M') whose lambdamax is blue-shifted by 60 nm from that of M became appreciable. The replacement abolished the shoulder of the ppRM spectrum. From these findings, we infer that the distance between the retinal and the 108-position in ppR is relatively long, and that in the M-state this distance is shortened.
Laboratory of Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, 060-0812, Japan.
K
K
Shimono
M
M
Iwamoto
M
M
Sumi
N
N
Kamo
D000595:Amino Acid Sequence; D019843:Archaeal Proteins; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics; D002338:Carotenoids; D004926:Escherichia coli; D025602:Halorhodopsins; D008715:Methionine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D025603:Sensory Rhodopsins; D017386:Sequence Homology, Amino Acid; D013057:Spectrum Analysis; D014633:Valine / Q000737:chemistry
D019843:Archaeal Proteins; D025602:Halorhodopsins; D025603:Sensory Rhodopsins; C061508:sensory rhodopsin II protein, archaeal; D002338:Carotenoids; D001436:Bacteriorhodopsins; D008715:Methionine; D014633:Valine