Effects of three characteristic amino acid residues of pharaonis phoborhodopsin on the absorption maximum.
Phoborhodopsin (pR or sensory rhodopsin II, sRII) or pharaonis phoborhodopsin (ppR or pharaonis sensory rhodopsin II, psRII) has a unique absorption maximum (lambda max) compared with three other archaeal rhodopsins: lambda max of pR or ppR at ca 500 nm and others at 560-590 nm. Alignment of amino acid sequences revealed three sites characteristic of the shorter wavelength-absorbing pigments. The amino acids of these three sites are conserved completely among archaeal rhodopsins having longer lambda max, and are different from those of pR or ppR. We replaced these amino acids of ppR with amino acids corresponding to those of bacteriorhodopsin, Val-108 to Met, Gly-130 to Ser and Thr-204 to Ala. The lambda max of V108M mutant was 502 nm with a slight redshift. G130S and T204A mutants had lambda max of 503 and 508 nm, respectively. Thus, each site contributes only a small effect to the color tuning. We then constructed three double mutants and one triple mutant. The opsin-shifts of these mutants suggest that Val-108 and Thr-204 or Gly-130 are synergistic, and that Gly-130 and Thr-204 work additively. Even in the triple mutant, the lambda max was 515 nm, an opsin-shift only ca 30% of the shift value from 500 to 560 nm. This means that there is another yet unidentified factor responsible for the color tuning.
Laboratory of Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
K
K
Shimono
M
M
Iwamoto
M
M
Sumi
N
N
Kamo
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D019843:Archaeal Proteins; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects; D002338:Carotenoids; D025602:Halorhodopsins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019633:Natronobacterium / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects; D010777:Photochemistry; D011487:Protein Conformation; D025603:Sensory Rhodopsins; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry
D000596:Amino Acids; D019843:Archaeal Proteins; D025602:Halorhodopsins; D025603:Sensory Rhodopsins; C061508:sensory rhodopsin II protein, archaeal; D002338:Carotenoids; D001436:Bacteriorhodopsins