Chemical Kinetic Analysis of Thermal Decay of Rhodopsin Reveals Unusual Energetics of Thermal Isomerization and Hydrolysis of Schiff Base

Abstract

The thermal properties of rhodopsin, which set the threshold of our vision, have long been investigated, but the chemical kinetics of the thermal decay of rhodopsin has not been revealed in detail. To understand thermal decay quantitatively, we propose a kinetic model consisting of two pathways: 1) thermal isomerization of 11-cis-retinal followed by hydrolysis of Schiff base (SB) and 2) hydrolysis of SB in dark state rhodopsin followed by opsin-catalyzed isomerization of free 11-cis-retinal. We solve the kinetic model mathematically and use it to analyze kinetic data from four experiments that we designed to assay thermal decay, isomerization, hydrolysis of SB using dark state rhodopsin, and hydrolysis of SB using photoactivated rhodopsin. We apply the model to WT rhodopsin and E181Q and S186A mutants at 55°C, as well as WT rhodopsin in H 2 O and D 2 O at 59°C. The results show that the hydrogen-bonding network strongly restrains thermal isomerization but is less important in opsin and activated rhodopsin. Furthermore, the ability to obtain individual rate constants allows comparison of thermal processes under various conditions. Our kinetic model and experiments reveal two unusual energetic properties: the steep temperature dependence of the rates of thermal isomerization and SB hydrolysis in the dark state and a strong deuterium isotope effect on dark state SB hydrolysis. These findings can be applied to study pathogenic rhodopsin mutants and other visual pigments.The dim light photoreceptor rhodopsin has been extensively studied over the past few decades (1-4). Rhodopsin consists of the seven-helical transmembrane opsin apoprotein covalently bound via a protonated Schiff base (SB) 5 to a chromophore, 11-cis-retinal, which isomerizes to all-trans-retinal upon absorption of a photon (5-7). The isomerization of retinal in the sterically crowded binding site of rhodopsin triggers conformational changes in the protein, leading to the active state, metarhodopsin II (Meta II) (8, 9). Meta II then activates the G protein transducin, setting off the visual signaling cascade (10).Although rhodopsin is known to have single-photon sensitivity, more photons are required to elicit a sensation of light in humans (11). Visual sensitivity can be limited by two factors that cause aberrant, false visual signaling: discrete dark noise and constitutive activation of rhodopsin. Discrete dark noise was first observed in 1972 by Srebro and Behbehani (12), who recorded electrophysiological signals generated by thermally induced chemical changes of visual pigments. The signals were identical to those generated by single photons. Baylor and coworkers (11,(13)(14)(15) further demonstrated that discrete dark noise of rhodopsin originates from spontaneous thermal isomerization of 11-cis-retinal. On the other hand, in some rhodopsin mutants, the SB linking retinal to opsin hydrolyzes, resulting in opsin that can constitutively activate transducin, causing partial or complete saturation of the rod response and desensitizing dim light visi...

Unusual kinetics of thermal decay of dim-light photoreceptors in vertebrate vision

“…Fig. 2 E and F shows the Arrhenius plots for thermal isomerization and hydrolysis of the PSB, which are the two competing reactions responsible for thermal decay (12,13) (Fig. 2D).…”

“…Fig. 2A shows the time-dependent UV-visible spectra of our expressed and purified bovine rhodopsin in 0.1% n-dodecyl-β-Dmaltoside (DDM) after being added to a preheated buffer that initiates the thermal decay, as in previous studies (12,13). The optical density at 500 nm (OD 500 ) decreases, whereas absorption at 380 nm (OD 380 ) increases, due to formation of all-trans retinyl Significance For vertebrates to have sensitive vision in dim light, any background signals in the dark must be minimal, i.e., thermal reactions of the visual pigment rhodopsin must be very slow.…”

Mice with a D190N Mutation in the Gene Encoding Rhodopsin: A Model for Human Autosomal-Dominant Retinitis Pigmentosa

“…However, our immunohistochemistry results show that rhodopsin was localized in the rod outer segments, not in any other layers, in wt and D190N/+ mice, suggesting that protein trafficking was normal in heterozygotes. In light of the desensitization seen on the scotopic ERG intensity series, the rhodopsin localization data may indicate that the D190N mutation renders rhodopsin thermally unstable (24,25), causing constitutive GPCR signaling and thereby maintaining a perpetual lightadapted state in the retina, in which case low intracellular Ca 2+ levels may be the cause of degeneration, not the UPR. However, although this is an exciting possibility, the data do not yet confirm such a conclusion and further investigation is necessary.…”

Structural role of the T94I rhodopsin mutation in congenital stationary night blindness

“…These thermal-shift measurements indicate an overall lower stability of the dark state for the two CSNB mutations compared to the WT. We investigated whether this effect is specifically due to disturbance of SB integrity by measuring the rate of thermal decay as a function of the decrease in absorption from retinal with protonated SB (Fig 2A-D Decay of the dark state involves two simultaneous spontaneous processes, that is, thermal isomerization of 11-cis to all-trans retinal and SB hydrolysis [27,28]. We measured the rate of thermal isomerization by HPLC analysis after extracting retinal after various times of incubation at 55°C (Fig 2E-H).…”