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

Abstract

A method was developed to measure Fouriertransform infrared (FTIR) difference spectra of detergentsolubilized 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), 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 fl-like species with maximal absorption at 380 nm that was able to activate guanine nucleotide exchange by btanducin. Rhodopsin versus metarhodopsin iH-like photoproduct FTIR-difference spectra were recorded for each sample. The COS-ceil rhodopsin and mutant difference spectra showed close correspondence to that of rhodopsin from disc membranes.Difference bands (rhodopsin/metarhodopsin II) at 1767/1750 cm'i and at 1734/1745 cm-' 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 H, in agreement with the isotope effects observed in spectra measured in 2H20. A photoproduct band at 1712 cm-' was not affected by either single or double replacements at positions 83 and 122. We deduce that the 1712 cm-' band arises from the protonation of Glu-113 in metarhodopsin II. Rhodopsin is a member of the superfamily of seventransmembrane-helix, G protein-coupled receptors. The rhodopsin chromophore 11-cis-retinal is covalently bound to the protein via a protonated Schiff base linkage (1) to a lysine residue (Lys-296 in bovine rhodopsin) (2, 3). After photoisomerization of the chromophore, thermal relaxation leads to an active conformation, R*, which binds the G protein transducin and thereby couples photon absorption to the visual signal transduction cascade. It has been shown by chemical modifications of Lys-296 in bovine rhodopsin that the deprotonation of the Schiff base is a prerequisite for R* formation (4, 5). Spectroscopically, this state is designated metarhodopsin II (MII) and characterized by a visible absorption maximum (Am.) at 380 nm, indicative of the unprotonated Schiff base of all-trans-retinal. Biochemical studies (6-10) and resonance Raman spectroscopy (11) of recombinant rhodopsins have shown that the positive charge at the Schiff base nitrogen in rhodopsin is stabilized by Glu-113, which acts as a Schiff base counterion in the transmembrane domain of the opsin.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.To investigate the protonation states and possible protonation changes of membrane-embedded carboxyl groups in rhodopsin and its MII photoproduct, we have performed Fourier-transform i...

Single amino acid residue as a functional determinant of rod and cone visual pigments

“…After harvesting, the cell membrane was isolated by means of sucrose flotation methods (21), and the opsin was extracted with buffer E [0.75% (wt/vol) CHAPS͞1 mg/ml L-␣-phosphatidylcholine (from egg yolk)͞50 mM Hepes͞140 mM NaCl͞1 mM DTT͞1 g/ml aprotinin͞1 g/ml leupeptin, pH 6.5 at 4ЊC]. The absorption maxima of the expressed rhodopsins were 507 (wild type), 491 (Q64K͞E122Q͞E150A), 508 (Q64K), 504 (E150A), 486 (E122Q), 504 (E122I), and 487 (E122D) nm, the latter three of which were in good agreement with those reported previously in bovine rhodopsin system (21)(22)(23)(24)(25)(26)(27). The maxima of the expressed chicken green were 511 (wild type) and 525 (cG-Q122E) nm, the former of which is similar to that reported previously (28).…”

Generation of a homology model of the human histamine H3 receptor for ligand docking and pharmacophore-based screening

“…The final model was submitted to pK a -shift calculations using the program UHBD [32], which suggested that residue D2.50 was -in analogy to D2.50 in bovine rhodopsin-in its protonated state [25].…”

“…Different to the model of Sippl et al which suggested an indirect influence of amino-acids varying between species, in the model of Yao antagonists made a direct contact to those residues, resulting in a ligand placement extending from D3.32 orthogonal to the membrane plane down to residue D2.50 [25,26]. In 2005, a model of the rat H 3 R was published by Lorenzi et al which was used to guide the successful design of further imidazole-containing H 3 R compounds [27].…”

Spectral Tuning in Visual Pigments: An ONIOM(QM:MM) Study on Bovine Rhodopsin and its Mutants

“…The overall charge of the system after adding missing hydrogen atoms (total of 5632 atoms) is +1 due to protonated Schiff-base (PSB) linkage between the 11-cis-retinal and Lys296, compensated by the negatively charged Glu113 counterion. [33][34][35] Although β-ionone ring orientation is 6-s-cis at the C 6 -C 7 bond of the polyene chain of the chromophore in all X-ray studies, 29,36-38 NMR studies give conflicting results: 6-s-trans in refs 39 and 40 ; 6-s-cis in refs 41-43 ; 6-s-cis (6-s-trans) with a 74% (26%) component in ref 44 . Low-level semiempirical MNDO-CI 45 and DFTB 46 calculations estimate 6-s-trans geometry more stable.…”