The G-protein-coupled receptor rhodopsin is activated by photoconversion of its covalently bound ligand 11-cis-retinal to the agonist all-trans-retinal. After lightinduced isomerization and early photointermediates, the receptor reaches a G-protein-dependent equilibrium between active and inactive conformations distinguished by the protonation of key opsin residues. In this report, we study the role of the 9-methyl group of retinal, one of the crucial steric determinants of light activation. We find that when this group is removed, the protonation equilibrium is strongly shifted to the inactive conformation. The residually formed active species is very similar to the active form of normal rhodopsin, metarhodopsin II. It has a deprotonated Schiff base, binds to the retinal G-protein transducin, and is favored at acidic pH. Our data show that the normal proton transfer reactions are inhibited in 9-demethyl rhodopsin but are still mandatory for receptor activation. We propose that retinal and its 9-methyl group act as a scaffold for opsin to adjust key proton donor and acceptor side chains for the proton transfer reactions that stabilize the active conformation. The mechanism may also be applicable to related receptors and may thus explain the partial agonism of certain ligands.The retinal photoreceptor rhodopsin is one of the archetypes of the G-protein-coupled receptor superfamily. It is composed of the apoprotein opsin comprising seven transmembrane helices (TMs) 1 and the chromophore 11-cis-retinal, which is covalently bound to Lys 296 in TM7 via a protonated Schiff base (SB), keeps the receptor in the inactive conformation, and acts as a highly effective inverse agonist. Following the absorption of a photon, the retinal isomerizes to a strained all-trans-conformation (1), which induces a series of conformational rearrangements of the opsin moiety. The final product of this reaction sequence is the active conformation (R*) that contains all-trans-retinal as a covalently bound agonist and is capable of catalyzing the activation of the retinal G-protein transducin (G t ) (for reviews see Refs. 2-4).These events can be summarized as shown in Fig. 1A). After cis-trans-isomerization and initial, short lived intermediates, illuminated rhodopsin progresses from the lumirhodopsin form to the first long lived state, metarhodopsin I (meta I). Up to and including meta I, the SB of the pigment remains protonated. Deprotonation of the SB and protonation of its counterion Glu 113 mark the transition from the meta I ( max ϭ 478 nm) to the metarhodopsin II (meta II) conformation (5), which is characterized by a strongly blue-shifted absorbance maximum ( max ϭ 380 nm). SB deprotonation is known to be necessary for interaction with G t (6 -8) and is accompanied by the uptake of a proton from the aqueous solution (9 -11). pH rate profiles of G t activation and proton uptake measurements have shown the opsin residue Glu 134 to be an important mediator of the uptake or even the acceptor of this proton (12, 13). Based on computer ...
The bacterial proton pump bacteriorhodopsin (BR) is a 26.5 kDa seven-transmembrane helical protein. Several structural models have been published at > or =1.55 A resolution. The initial cis-trans isomerization of the retinal moiety involves structural changes within <1 A. To understand the chromophore-protein interactions that are important for light-driven proton transport, very accurate measurements of the protein geometry are required. To reveal more structural details at the site of the retinal, we have, therefore, selectively labeled the tryptophan side chains of BR with (15)N and metabolically incorporated retinal, (13)C-labeled at position 14 or 15. Using these samples, heteronuclear distances were measured with high accuracy using SFAM REDOR magic angle spinning solid-state NMR spectroscopy in dark-adapted bacteriorhodopsin. This NMR technique is applied for the first time to a high-molecular mass protein. Two retinal conformers are distinguished by their different isotropic 14-(13)C chemical shifts. Whereas the C14 position of 13-cis-15-syn-retinal is 4.2 A from [indole-(15)N]Trp86, this distance is 3.9 A in the all-trans-15-anti conformer. This latter distance allows us to check on the details of the active center of BR in the various published models derived from X-ray and electron diffraction data. The experimental approach and the results reported in this paper enforce the notion that distances between residues of a membrane protein binding pocket and a bound ligand can be determined at subangstrom resolution.
A series of vitamin-A aldehydes (retinals) with modified alkyl group substituents (9-demethyl-, 9-ethyl-, 9-isopropyl-, 10-methyl, 10-methyl-13-demethyl-, and 13-demethyl retinal) was synthesized and their 11-cis isomers were used as chromophores to reconstitute the visual pigment rhodopsin. Structural changes were selectively introduced around the photoisomerizing C11=C12 bond. The effect of these structural changes on rhodopsin formation and bleaching was determined. Global fit of assembly kinetics yielded lifetimes and spectral features of the assembly intermediates. Rhodopsin formation proceeds stepwise with prolonged lifetimes especially for 9-demethyl retinal (longest lifetime τ3 = 7500 s, cf., 3500 s for retinal), and for 10-methyl retinal (τ3 = 7850 s). These slowed-down processes are interpreted as either a loss of fixation (9dm) or an increased steric hindrance (10me) during the conformational adjustment within the protein. Combined quantum mechanics and molecular mechanics (QM/MM) simulations provided structural insight into the retinal analogues-assembled, full-length rhodopsins. Extinction coefficients, quantum yields and kinetics of the bleaching process (μs-to-ms time range) were determined. Global fit analysis yielded lifetimes and spectral features of bleaching intermediates, revealing remarkably altered kinetics: whereas the slowest process of wild-type rhodopsin and of bleached and 11-cis retinal assembled rhodopsin takes place with lifetimes of 7 and 3.8 s, respectively, this process for 10-methyl-13-demethyl retinal was nearly 10 h (34670 s), coming to completion only after ca. 50 h. The structural changes in retinal derivatives clearly identify the precise interactions between chromophore and protein during the light-induced changes that yield the outstanding efficiency of rhodopsin.
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