Protonation states of membrane-embedded carboxylic acid groups in rhodopsin and metarhodopsin II: a Fourier-transform infrared spectroscopy study of site-directed mutants.
A method was developed to measure Fourier-transform infrared (FTIR) difference spectra of detergent-solubilized rhodopsin expressed in COS cells. Experiments were performed on native bovine rhodopsin, rhodopsin expressed in COS cells, and three expressed rhodopsin mutants with amino acid replacements of membrane-embedded carboxylic acid groups: Asp-83-->Asn (D83N), Glu-122-->Gln (E122Q), and the double mutant D83N/E122Q. Each of the mutant opsins bound 11-cis-retinal to yield a visible light-absorbing pigment. Upon illumination, each of the mutant pigments formed a metarhodopsin II-like species with maximal absorption at 380 nm that was able to activate guanine nucleotide exchange by transducin. Rhodopsin versus metarhodopsin II-like photoproduct FTIR-difference spectra were recorded for each sample. The COS-cell rhodopsin and mutant difference spectra showed close correspondence to that of rhodopsin from disc membranes. Difference bands (rhodopsin/metarhodopsin II) at 1767/1750 cm-1 and at 1734/1745 cm-1 were absent from the spectra of mutants D83N and E122Q, respectively. Both bands were absent from the spectrum of the double mutant D83N/E122Q. These results show that Asp-83 and Glu-122 are protonated both in rhodopsin and in metarhodopsin II, in agreement with the isotope effects observed in spectra measured in 2H2O. A photoproduct band at 1712 cm-1 was not affected by either single or double replacements at positions 83 and 122. We deduce that the 1712 cm-1 band arises from the protonation of Glu-113 in metarhodopsin II.
Fahmy
K
K
Howard Hughes Medical Institute, Rockefeller University, New York, NY 10021.
Jäger
F
F
Beck
M
M
Zvyaga
T A
TA
Sakmar
T P
TP
Siebert
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine; D001224:Aspartic Acid; D002460:Cell Line; D005971:Glutamates; D018698:Glutamic Acid; D005973:Glutamine; D007700:Kinetics; D008027:Light; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012172:Retinaldehyde / Q000378:metabolism; D012243:Rhodopsin / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism*; D013053:Spectrophotometry; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D013997:Time Factors; D014162:Transfection
D005971:Glutamates; D011994:Recombinant Proteins; D005973:Glutamine; D001224:Aspartic Acid; D018698:Glutamic Acid; C013110:metarhodopsins; D001216:Asparagine; D012243:Rhodopsin; D012172:Retinaldehyde