New tool for spectroscopists

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.

Proton transfer reactions in bacteriorhodopsin were investigated by Fourier transform infrared spectroscopy, using a mutant protein in which Asp-96 was replaced by Asn-96. By comparison of the BR - K, BR - L, and BR - M difference spectra (BR indicating bacteriorhodopsin ground state and K, L, and M indicating photo-intermediates) of the wild-type protein with the corresponding difference spectra of the mutant protein, detailed insight into the functional role of this residue in the proton pump mechanism is obtained. Asp-96 is protonated in BR, as well as another aspartic residue, which is tentatively assigned to be Asp-115. Asp-96 is not affected in the primary photoreaction. During formation of the L intermediate it is subjected to a change in the H-bonding character of its carboxylic group, but no deprotonation occurs at this reaction step. Also, in the mutant protein a light-induced structural change of the protein interior near the Asn-96 residue is probed. The BR - M difference spectrum of the mutant protein lacks the negative carbonyl band at 1742 cm-1 of Asp-96 and in addition a positive band at about 1378 cm-1, which is most likely to be caused by the carboxylate vibration of Asp-96. This argues for a deprotonation of Asp-96 in the time range of the M intermediate during its photostationary accumulation. On the basis of these results, it is suggested that the point mutation does not induce a gross change of the protein structure, but a proton-binding site in the proton pathway from the cytoplasmic side to the Schiff base is lost.