Rhodopsin with 11-cis-Locked Chromophore Is Capable of Forming an Active State Photoproduct

Abstract

The visual pigment rhodopsin is characterized by an 11-cis retinal chromophore bound to Lys-296 via a protonated Schiff base. Following light absorption the C 11 ‫؍‬C 12 double bond isomerizes to trans configuration and triggers protein conformational alterations. These alterations lead to the formation of an active intermediate (Meta II), which binds and activates the visual G protein, transducin. We have examined by UV-visible and Fourier transform IR spectroscopy the photochemistry of a rhodopsin analogue with an 11-cis-locked chromophore, where cis to trans isomerization around the C 11 ‫؍‬C 12 double bond is prevented by a 6-member ring structure (Rh 6.10 ). Despite this lock, the pigment was found capable of forming an active photoproduct with a characteristic protein conformation similar to that of native Meta II. This intermediate is further characterized by a protonated Schiff base and protonated Glu-113, as well as by its ability to bind a transducin-derived peptide previously shown to interact efficiently with native Meta II. The yield of this active photointermediate is pH-dependent and decreases with increasing pH. This study shows that with the C 11 ‫؍‬C 12 double bond being locked, isomerization around the C 9 ‫؍‬C 10 or the C 13 ‫؍‬C 14 double bonds may well lead to an activation of the receptor. Additionally, prolonged illumination at pH 7.5 produces a new photoproduct absorbing at 385 nm, which, however, does not exhibit the characteristic active protein conformation.Rhodopsin, a seven-transmembrane helical protein, is composed of 348 amino acids and a ligand, 11-cis retinal, which covalently binds to the protein through a protonated Schiff base linkage to the ⑀-amino group of Lys-296 in the center of helix 7 (1). Glu-113 at helix 3 serves as the counterion of the protonated Schiff base (2-4). The recently resolved crystal structure of rhodopsin (5) provides detailed structural information on the interactions between the retinal chromophore and its surrounding residues, which contribute to the red-shifted absorption maximum of the chromophore ( max 500 nm) relative to protonated retinal Schiff base in methanol solution ( max 440 nm) and to the high pK a of the protonated Schiff base (6). Following light absorption, the retinal chromophore isomerizes from 11-cis to trans configuration in 200 fs (7). A series of photointermediates are produced that can be trapped below characteristic transition temperatures (8, 9): bathorhodopsin ( max 543 nm, T Ͻ Ϫ140°C), lumirhodopsin ( max 497 nm, T Ͻ Ϫ40°C), and metarhodopsin I (Meta I, 1 max 478 nm) above Ϫ40°C, which equilibrates with metarhodopsin II (Meta II, max 380 nm). Meta II is the active species, capable of activating transducin, the visual G protein. The equilibrium between Meta I and Meta II is affected by temperature, pressure, pH, and glycerol (9), as well as by ions (10, 11).Meta II formation is associated with a movement of helix 6 relative to helix 3 (12, 13), a translocation of a proton from the Schiff base to its counterion, Glu-113, and re...

Photocyclic behavior of rhodopsin induced by an atypical isomerization mechanism

“…S1) have implied their ability to activate the G protein transducin (G t ). These results suggest an alternative mechanism that can propagate the downstream visual cascades (9)(10)(11)(12)(13)(14). In this study, we provide direct evidence that Rh6mr can activate G t by an atypical cis-to-dicis isomerization that is also photocyclic, undergoing an 11,13-dicis-to-11-cis reisomerization.…”

“…Although Rh6mr G t activation capability has been discussed previously (9)(10)(11)(12)(13)(14), its underlying molecular mechanism is poorly understood. In this study, we found that the 11,13-dicis 6mr isoform is generated as the photoproduct during illumination, and is exclusively responsible for the dynamic structural change in Rh6mr required to attain a Meta-II-like intermediate state that can activate G t .…”

“…2A). Previous FTIR studies show Glu113 to be protonated after light illumination of Rh6mr (14). Based on the combination of our spectral ( Fig.…”

A Nonbleachable Rhodopsin Analogue with a Slow Photocycle

“…In a previous attempt to examine Rh 6.10 with Fourier-transform infrared (FTIR) 1 spectroscopic techniques, however, no substantial amounts of a putative active state could be detected at neutral pH (7). In a recent study by our group, we were able to detect at more acidic pH the formation of such a photoproduct state, which showed important properties of an active Meta II-like state, namely the characteristic IR difference spectrum of Meta II, as well as binding to and stabilization by G protein-derived peptides (4). In contrast to native Meta II, however, the Schiff base in this photoproduct with active state conformation remained protonated, indicating a different positioning of the Schiff base within the protein compared with native rhodopsin.…”

“…3 and references in Ref. 4). Much attention had been drawn to 11-cis ring-constrained retinals with a 6-member ring, Re 6.10 , as they recombined with opsin to pigments that were capable of at least some rudimentary photochemistry and some marginal activity toward the visual G protein, transducin (5,6).…”