The master circadian oscillator in the hypothalamic suprachiasmatic nucleus is entrained to the day/night cycle by retinal photoreceptors. Melanopsin (Opn4), an opsin-based photopigment, is a primary candidate for photoreceptor-mediated entrainment. To investigate the functional role of melanopsin in light resetting of the oscillator, we generated melanopsin-null mice (Opn4-/-). These mice entrain to a light/dark cycle and do not exhibit any overt defect in circadian activity rhythms under constant darkness. However, they display severely attenuated phase resetting in response to brief pulses of monochromatic light, highlighting the critical role of melanopsin in circadian photoentrainment in mammals.
Rhodopsin is a member of the superfamily of G-protein-coupled receptors. This seven ␣-helix transmembrane protein is the visual pigment of the vertebrate rod photoreceptor cells that mediate dim light vision. In the active binding site of this protein the ligand or chromophore, 11-cis-retinal, is covalently bound via a protonated Schiff base to lysine residue 296. Here we present the complete 1 H and 13 C assignments of the 11-cis-retinylidene chromophore in its ligand-binding site determined with ultra high field magic angle spinning NMR. Native bovine opsin was regenerated with 99% enriched uniformly 13 C-labeled 11-cis-retinal. From the labeled pigment, 13 C carbon chemical shifts could be obtained by using two-dimensional radio frequency-driven dipolar recoupling in a solid-state magic angle spinning homonuclear correlation experiment.
The present study focuses on ligand-protein interactions in a rhodopsin analogue generated from bovine opsin and the 10-methyl homologue of 11-cis-retinal. The analogue pigment displays a reduced alpha-band at 506 +/- 2 and a stronger beta-band at 325 nm. Remarkably, the rotational strength of these bands observed in visible circular dichroism spectra was found to be similar for both native and 10-methyl rhodopsin. The quantum yield of the analogue pigment was determined to be 0.55. All photointermediates were analyzed by Fourier transform infrared difference spectroscopy. At the batho stage, strong hydrogen-out-of-plane vibrations were observed, indicating that the 10-methyl chromophore also adopts a distorted all-trans conformation at this stage. In contrast to native rhodopsin, the batho intermediate of the 10-methyl pigment is stable up to 180 K and only slowly decays to the next intermediate between 180 and 210 K. As in native rhodopsin, the 10-methyl metarhodopsin I intermediate is generated at about 220 K, but its transition to the metarhodopsin II state is again shifted to a much higher temperature (> 293 K) than for the native pigment (> 260 K). Infrared analysis, nevertheless, shows that the conformational changes in the photointermediates of the 10-methyl pigment are basically identical with those observed in the native pigment. This is supported by a signal function assay, showing that the analogue pigment is able to activate transducin. The dual effect of the 10-methyl group on the photocascade is attributed to steric interactions which, initially, hamper the relaxation of strain in the polyene chain of the chromophore and, eventually, interfere with the conformational rearrangements of the protein moiety required to adopt the active conformation of the receptor. Our data provide direct support for the concept that the relaxation of strain in the retinal polyene chain acts as the major driving force of the photocascade dark reaction.
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