Protonation States of Membrane-Embedded Carboxylic Acid Groups in Rhodopsin and Metarhodopsin II: A Fourier-Transform Infrared Spectroscopy Study of Site-Directed Mutants

• Published in: Proceedings of the National Academy of Sciences - PNAS 1993-11, Vol.90 (21), p.10206-10210
• Main Authors: Fahmy, Karim, Jager, Frank, Beck, Mareike, Zvyaga, Tatyana A., Sakmar, Thomas P., Siebert, Friedrich
• Format: Article
• Language: English
• Subjects: Amides; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Anatomy & physiology; Animals; Asparagine; Aspartic Acid; Biochemistry; Biological and medical sciences; Carboxyl compounds; Carboxylic acids; Cattle; Cell Line; Fundamental and applied biological sciences. Psychology; Glutamates; Glutamic Acid; Glutamine; Kinetics; Light; Mutagenesis, Site-Directed; Non metallic chromoproteins, photoproteins; Pigments; Protein Conformation; Proteins; Protons; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Retinaldehyde - metabolism; Rhodopsin - analogs & derivatives; Rhodopsin - chemistry; Rhodopsin - metabolism; Schiff bases; Scientific imaging; Spectral bands; Spectrophotometry; Spectroscopy, Fourier Transform Infrared - methods; Time Factors; Transfection; Vibration; Vibrational spectra
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.90.21.10206

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-1and at 1734/1745 cm-1were 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 in2H2O. A photoproduct band at 1712 cm-1was not affected by either single or double replacements at positions 83 and 122. We deduce that the 1712 cm-1band arises from the protonation of Glu-113 in metarhodopsin II.