Protonation states of membrane-embedded carboxylic acid groups in rhodopsin and metarhodopsin II: a Fourier-transform infrared spectroscopy study of site-directed mutants. Export

@article{citeulike:4197375, abstract = {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.}, author = {Fahmy, K. and J\"{a}ger, F. and Beck, M. and Zvyaga, T. A. and Sakmar, T. P. and Siebert, F.}, citeulike-article-id = {4197375}, citeulike-linkout-0 = {http://view.ncbi.nlm.nih.gov/pubmed/7901852}, citeulike-linkout-1 = {http://www.hubmed.org/display.cgi?uids=7901852}, comment = {Evidence that D2.50 is protonated both in the inactive and MII states of rhodopsin}, day = {1}, issn = {0027-8424}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, keywords = {activation, d250, protonation, rhodopsin, spectroscopy, switch}, month = {November}, number = {21}, pages = {10206--10210}, posted-at = {2009-03-20 11:48:33}, priority = {2}, title = {Protonation states of membrane-embedded carboxylic acid groups in rhodopsin and metarhodopsin II: a Fourier-transform infrared spectroscopy study of site-directed mutants.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/7901852}, volume = {90}, year = {1993} }

TY - JOUR ID - citeulike:4197375 L3 - citeulike-article-id:4197375 N2 - 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. IS - 21 JF - Proceedings of the National Academy of Sciences of the United States of America SN - 0027-8424 EP - 10210 TI - Protonation states of membrane-embedded carboxylic acid groups in rhodopsin and metarhodopsin II: a Fourier-transform infrared spectroscopy study of site-directed mutants. VL - 90 SP - 10206 KW - activation KW - d250 KW - protonation KW - rhodopsin KW - spectroscopy KW - switch N1 - Evidence that D2.50 is protonated both in the inactive and MII states of rhodopsin AU - Fahmy, K AU - Jäger, F AU - Beck, M AU - Zvyaga, TA AU - Sakmar, TP AU - Siebert, F PY - 1993/11/01/ UR - http://view.ncbi.nlm.nih.gov/pubmed/7901852 ER -