Photocyclic behavior of rhodopsin induced by an atypical isomerization mechanism

Abstract

Vertebrate rhodopsin (Rh) contains 11-cis-retinal as a chromophore to convert light energy into visual signals. On absorption of light, 11-cis-retinal is isomerized to all-trans-retinal, constituting a one-way reaction that activates transducin (G_t) followed by chromophore release. Here we report that bovine Rh, regenerated instead with a six-carbon-ring retinal chromophore featuring a C¹¹=C¹² double bond locked in its cis conformation (Rh6mr), employs an atypical isomerization mechanism by converting 11-cis to an 11,13-dicis configuration for prolonged G_t activation. Time-dependent UV-vis spectroscopy, HPLC, and molecular mechanics analyses revealed an atypical thermal reisomerization of the 11,13-dicis to the 11-cis configuration on a slow timescale, which enables Rh6mr to function in a photocyclic manner similar to that of microbial Rhs. With this photocyclic behavior, Rh6mr repeatedly recruits and activates G_t in response to light stimuli, making it an excellent candidate for optogenetic tools based on retinal analog-bound vertebrate Rhs. Overall, these comprehensive structure-function studies unveil a unique photocyclic mechanism of Rh activation by an 11-cis-to-11,13-dicis isomerization.

Keywords: GPCR; chromophore; isomerization; rhodopsin; vision.

Fig. 1.

G_t activation kinetics of Rh6mr. (A) The G_t activation ability of Rh6mr was monitored by an increase in the intrinsic tryptophan fluorescence of the G_t alpha subunit in the presence of light-activated Rh6mr at 20 °C (pH 7.0). (B) Arrhenius plot for Rh6mr G_t activation for a temperature range of 8 to 20 °C. Natural logarithms of the measured G_t activation rate constants were plotted against inverse temperatures. The slopes of Arrhenius lines (Rh6mr, solid red; Rh, black dashes) provide the energy required for the Rh6mr conformational change needed for G_t activation (E_a).

Fig. 2.

Photocyclic behavior and photosensitivity of Rh6mr. (A) Time-dependent UV-vis absorption spectra of Rh6mr in the dark and over a period of 20 h after a 1-min illumination at pH 7.0 at 20 °C. (A, Inset) Expanded absorption spectra of Rh6mr in the dark and immediately after a 1-min illumination. (B) The photosensitivity of Rh6mr and Rh. The percentage of residual pigment was plotted on a semilogarithmic scale against the incident photon flux and fitted with an exponential function (the Rh6mr photosensitivity plot is scaled down to show the comparison with Rh). The photosensitivity (ϕ) of Rh6mr (solid red), estimated by the slope of the fitting line, was 0.043 ± 0.0004 relative to that of Rh (black dashes). (C) Energy plot of Rh docked with the 11-cis 6mr isomer during the preferred anticlockwise rotation of the 6mr C¹²-C¹³-C¹⁴-C¹⁵ dihedral angle. The energy after a half-rotation (label II) is higher than the initial minimum at 170° (label 0), suggesting the plausibility of the 11,13-dicis–to–11-cis reisomerization. Waters 2a and 2b are shown as purple spheres, and hydrogen bonds are shown as dashed green sticks.

Fig. 3.

Time-dependent HPLC analyses revealing prolonged G_t activation of Rh6mr. (A) Time-dependent HPLC analysis of photoactivated Rh6mr was performed over a 48-h period. Rh6mr was kept at 20 °C in the dark for either 24 or 48 h after a 1-min illumination with 480- to 520-nm light. Extraction of retinal-oximes from Rh6mr was performed as described in Materials and Methods. (A, Inset) Overlay of time-dependent HPLC chromatograms of peak 3 (11-cis 6mr isomer) showing reisomerization of the 11,13-dicis to the 11-cis form. (B) Comparison of G_t activation efficiencies of Rh6mr at different time points within a 24-h period. Samples were kept at 20 °C in the dark for 1, 1.5, 2.0, and 24 h after a 1-min illumination with 480- to 520-nm light at pH 7.0. Fluorescence intensities were monitored (dotted lines) and fitted by single-exponential functions (solid lines). (C) Prolonged and cyclic G_t activation of Rh6mr. The G_t activation rate constants of Rh6mr samples at time points 0, 1, 1.5, 2, and 24 h after illumination were plotted against time. Independent 24-h samples were subjected to an additional illumination, and the ensuing G_t activation rates were plotted similarly. Closed and open squares correspond to the experimental and extrapolated G_t activation rates, respectively (Rh6mr, red; Rh, black). (D) HPLC chromatograms of Rh6mr regenerated with pure 11,13-dicis and 11-cis 6mr isomers. (D, Top) HPLC analysis under dark (black) and light (red) conditions revealed that the 11,13-dicis isomer is converted exclusively to the 11-cis isomer after binding with the opsin protein moiety. (D, Bottom) Illustration showing the photocyclic behavior of Rh6mr regenerated with pure 11,13-dicis and 11-cis 6mr isomers. RT, room temperature.

Fig. 4.

Preferential configuration of 6mr in Rh6mr crystals. (A) Crystal structure of Rh6mr. The retinal-binding pocket of Rh6mr shows a 2F_o-F_c density (blue mesh) corresponding to 6mr occupying the same binding pocket as 11-cis-retinal in Rh. The 2F_o-F_c density contoured at 1.1σ fits well with the 11-cis 6mr isomer covalently linked to Lys296. The 2F_o-F_c density map was B factor-sharpened (B_sharp = −178 Ų). (B) HPLC analysis of washed Rh6mr crystals showing the 11-cis 6mr isomer (peak 3) as the predominant configuration in the retinal-binding pocket. Rh6mr crystals were obtained by treating bleached ROS membranes with a 6mr isomer mixture. Five or six Rh6mr crystals (>100 μm in their longest dimension) were washed three times in 100 μL reservoir solution followed by extraction of 6mr-oximes and HPLC analysis.

Fig. 5.

Proposed model for G_t activation by Rh6mr upon illumination. (A) In Rh, absorption of a photon leads to retinal cis–trans isomerization. This causes a conformational change in the opsin protein moiety, culminating in the formation of its activated Meta-II state. Upon Meta-II formation, the proton from the retinal Schiff base dissociates, disturbing the intramolecular hydrogen-bonding network in the retinal-binding pocket. This causes a conformational change, opening the cytoplasmic side and enabling G_t to interact with Meta-II. (B) Alternatively, in Rh6mr, the 11-cis 6mr isomer is isomerized to its 11,13-dicis form upon activation by a light stimulus, causing a helical structural change in the Rh6mr opsin moiety forming a Meta-II–like state. Unlike Rh, inactive Rh6mr has a relatively open cytoplasmic side, allowing bulk solvent and hydroxylamine to gain access to the protonated Schiff base. Upon light activation, the cytoplasmic side of Rh6mr undergoes additional opening that enables G_t to interact with the cytoplasmic surface of Rh6mr. Interestingly, activated Rh6mr can thermally revert back to its inactive form in a slow photocyclic manner.