Chromophore structural changes in rhodopsin from nanoseconds to microseconds following pigment photolysis

Abstract

Rhodopsin is a prototypical G proteincoupled receptor that is activated by photoisomerization of its 11-cis-retinal chromophore. Mutant forms of rhodopsin were prepared in which the carboxylic acid counterion was moved relative to the positively charged chromophore Schiff base. Nanosecond time-resolved laser photolysis measurements of wild-type recombinant rhodopsin and two mutant pigments then were used to determine reaction schemes and spectra of their early photolysis intermediates. These results, together with linear dichroism data, yielded detailed structural information concerning chromophore movements during the first microsecond after photolysis. These chromophore structural changes provide a basis for understanding the relative movement of rhodopsin's transmembrane helices 3 and 6 required for activation of rhodopsin. Thus, early structural changes following isomerization of retinal are linked to the activation of this G protein-coupled receptor. Such rapid structural changes lie at the heart of the pharmacologically important signal transduction mechanisms in a large variety of receptors, which use extrinsic activators, but are impossible to study in receptors using diffusible agonist ligands.Rhodopsin is the light-triggered membrane receptor in rod outer segments responsible for dim-light vision. Absorption of light transforms rhodopsin (rho) into an activated state, which interacts with a heterotrimeric G protein to initiate an enzyme cascade leading to visual transduction. To reach this active state, called metarhodopsin (meta) II (1, 2), rho passes through a number of intermediates that have been characterized by UV-visible, Fourier-transform infrared (FTIR), Raman, and NMR spectroscopies, in addition to spin-labeling and biochemical studies (3-10). One way to characterize the intermediates is to trap them at low temperatures. This approach led to the classical ''bleaching'' scheme ( max values in nanometers): rho (498) 3 bathorhodopsin (batho) (542) 3 lumirhodopsin (lumi) (497) 3 meta I (480) 3 meta II (380) Scheme I However, at more physiologically relevant temperatures, time-resolved measurements up to 1 msec after photolysis reveal a new intermediate and a different kinetic scheme (3, 11). The following reaction scheme was proposed to occur during the nanosecond-to-microsecond time regime (3): rho (498) 3 batho (529) ª bsi (477) 3 lumi (492) 3 . . . Scheme II The new intermediate, blue-shifted intermediate of rho (bsi), is entropically favored and thus not trapped at low temperatures (11).The role of various structural features of the retinal chromophore in early rho photointermediates has been studied through time-resolved spectral measurements of artificial rho, in which the native chromophore is replaced by synthetically modified retinals (12). In studies of bacteriorhodopsin, timeresolved absorption spectroscopy has been shown to be particularly valuable when combined with site-directed mutagenesis to probe the roles of specific protein residues in different intermediates (13). Unti...

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

“…The nuclear spin interactions in solid-state 2 H NMR 24 as well as the electronic transition dipole moment 46 transform by the totally symmetric representation of the D ∞h point group. They are invariant to the symmetry operations of inversion, rotation, or reflection as found in standard character tables.…”

“…Analogous electronic transition dipole (TD) orientations are denoted by and θ TD . The Ci-Ck bond angle to the local membrane normal is , and is calculated from the C9 methyl the orientation, together with the transition dipole in either the dark 44,46 or meta I 43 state. According to Eq.…”

“…The three molecular planes share two bonds in common, so a total of four independent parameters (degrees of freedom) is needed to define the retinal structure. Since data for the C1 methyl group are unavailable, the electronic transition dipole moment of the retinylidene chromophore from linear dichroism measurements is introduced as a fourth orientational restraint [43][44][45][46] . Circular dichroism (CD) data are also available for the dark state of rhodopsin, which yield information on the enantiomeric selectivity of the protein binding pocket 33,34 .…”

Computational Studies of the Primary Phototransduction Event in Visual Rhodopsin

“…In particular, NMR studies by Watts and co-workers, 41 as well as theoretical work by Birge and coworkers, 42 concluded that the chromophore geometry is 6s-trans at the C6-C7 bond, in contrast to the 6s-cis conformation assumed by earlier NMR and optical studies. 33,43,[45][46][47][48][49][50] To address the orientation of the -ionone ring, according to rigorous DFT QM/MM calculations, a computational model of the 6s-trans isomer has been constructed by geometry relaxation of the system after rotation of the -ionone ring around the C6-C7 bond. 2 The resulting 6s-trans structure, obtained at the ONIOM electronicembedding (B3LYP/6-31G*:Amber) level of theory, was found to be as stable as the 6s-cis isomer for many thermally accessible configurations and about 8 kcal/mol less stable than the 6s-cis structure when minimum energy geometries are compared.…”

Dynamic Structure of Retinylidene Ligand of Rhodopsin Probed by Molecular Simulations

“…Remarkably, upon absorbing a photon it instantly isomerizes (within 200 fs 17,22 ) to the all-trans state, and subsequently becomes an agonist 13,14 . The protein relaxes through a series of spectroscopically defined intermediates 23 until it reaches the MI state, which exists in equilibrium with the active MII state 13,14,16,18 . The central role of retinal in directing this relaxation and promoting MII formation has been extensively investigated by a number of approaches.…”