An Additional Methyl Group at the 10-Position of Retinal Dramatically Slows down the Kinetics of the Rhodopsin Photocascade

Abstract

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.

Structural Analysis and Dynamics of Retinal Chromophore in Dark and Meta I States of Rhodopsin from 2H NMR of Aligned Membranes

“…Twisting of the retinal molecule obtained from the relative 2 H NMR tensor orientations 27 is in agreement with other biophysical studies 14,23,27,[29][30][31]34,46,52,[55][56][57][58][59][60] , and is key to understanding its photoreaction dynamics 32,[61][62][63] . Of particular significance for rhodopsin is the nonplanarity of the conjugated polyene chain of retinal, together with the β-ionone ring 23 .…”

“…This approach is analogous to solution NMR, where residual dipolar couplings are used with distance restraints from nuclear Overhauser effect (NOE) spectroscopy. A highly distorted structure is found for retinal in the dark state, consistent with vibrational 9,[30][31][32] and circular dichroism (CD) spectroscopy 30,31 , and with bioorganic studies of rhodopsin 33,34 . Despite constraints of the binding cavity the ligand undergoes restricted motion, as shown by the rapidly spinning methyl groups, with significant off-axial fluctuations.…”

“…Of particular significance for rhodopsin is the nonplanarity of the conjugated polyene chain of retinal, together with the β-ionone ring 23 . Nonplanar distortion of the retinal ligand is also shown by the presence of hydrogen-out-of-plane (HOOP) modes in resonance Raman 32,62,64,65 and Fourier transform infrared 56 spectroscopy, and by CD studies 30,31,34,55 . Compared to previous 2 H NMR studies 38,41 and quantum chemical calculations 19 , we obtain different results for the retinylidene methyl group orientations that are consistent with X-ray crystallographic data 14,17 .…”

“…Compared to previous 2 H NMR studies 38,41 and quantum chemical calculations 19 , we obtain different results for the retinylidene methyl group orientations that are consistent with X-ray crystallographic data 14,17 . All of these aspects are significant for explaining how the photonic energy is stored, released, and focused or dissipated in the triggering of visual signaling by rhodopsin in photoreceptor membranes 19,30,31,34,59,60 .…”

1 H and 13 C MAS NMR evidence for pronounced ligand–protein interactions involving the ionone ring of the retinylidene chromophore in rhodopsin

“…Furthermore, the observed upfield ⌬ lig C ϭ Ϫ2.5 ppm of CH 3 -20 is in line with a distorted s-trans conformation of the 12-s bond. Addition of a methyl group at C-10, giving 10-methylrhodopsin, yields a larger out-of-plane distortion in the isomerization region compared with rhodopsin and an additional upfield ⌬ lig C ϭ Ϫ1.6 ppm compared with the C-20 response in rhodopsin (10,45,46). This finding suggests a correlation between torsion and the 13 C-20 shift.…”