Resonance Raman Analysis of the Mechanism of Energy Storage and Chromophore Distortion in the Primary Visual Photoproduct

Abstract

The vibrational structure of the chromophore in the primary photoproduct of vision, bathorhodopsin, is examined to determine the cause of the anomalously decoupled and intense C 11 =C 12 hydrogenout-of-plane (HOOP) wagging modes and their relation to energy storage in the primary photoproduct. Low-temperature (77 K) resonance Raman spectra of Glu181 and Ser186 mutants of bovine rhodopsin reveal only mild mutagenic perturbations of the photoproduct spectrum suggesting that dipolar, electrostatic, or steric interactions with these residues do ded by NIHnot cause the HOOP mode frequencies and intensities. Density functional theory calculations are performed to investigate the effect of geometric distortion on the HOOP coupling. The decoupled HOOP modes can be simulated by imposing ∼40° twists in the same direction about the C 11 =C 12 and C 12 -C 13 bonds. Sequence comparison and examination of the binding site suggests that these distortions are caused by three constraints consisting of an electrostatic anchor between the protonated Schiff base and the Glu113 counterion, as well as steric interactions of the 9-and 13-methyl groups with surrounding residues. This distortion stores light energy that is used to drive the subsequent protein conformational changes that activate rhodopsin.Photoactive proteins play a vital role in a variety of light-energy and light-signaling processes that are characterized by pico-to femtosecond light-driven structural changes of a proteinbound chromophore followed by activated conformational changes of the protein. In systems such as rhodopsin (Rho) 1 (1), bacteriorhodopsin (2,3), halorhodopsin (4), photoactive yellow protein (5), and phytochrome (6,7), primary high-energy intermediates are produced with structurally perturbed chromophores. What is the nature of the protein-chromophore interactions that store the energy needed to drive subsequent protein conformational changes? To address this question, we have studied the photoactivation of rhodopsin, a dim-light photoreceptor, using a multidisciplinary approach that integrates Raman spectroscopy, mutagenesis, density functional theory (DFT) calculations, and bioinformatic analysis. † Funding was provided by NIH Grant EY02051 (to R.A.M. Rho is a paradigmatic G protein-coupled receptor, whose 7-transmembrane helical structure forms a binding pocket for 11-cis-retinal, which is covalently linked to Lys296 on helix 7. Within 200 fs after photoexcitation (8,9), the 11-cis-retinal protonated Schiff base (PSB) isomerizes to all-trans-retinal (ATR) with a quantum yield of 0.65 to form the primary photoproduct, Bathorhodopsin (10,11), which stores ∼30 kcal/mol or ∼60% of the incident photon energy (Figure 1) (12)(13)(14). Subsequently, the highly distorted chromophore relaxes, and the transduced energy is used to drive conformational changes in the opsin protein (15)(16)(17)(18). Recently, we found that this structural evolution includes a retinal counterion switch from Glu113 in the dark state to Glu181 in the metarhodopsin I (Met...

A large geometric distortion in the first photointermediate of rhodopsin, determined by double-quantum solid-state NMR

“…These calculations are clearly only indicative since the whole protein has been neglected. Nevertheless, more sophisticated quantum mechanical calculations do agree with cis-trans isomerization of the protein-bound chromophore leading to a highly strained all-trans-conformer in bathorhodopsin (Gascon et al 2006;Bifone et al 1997;Frutos et al 2007;Polli et al 2010;Schapiro et al 2011;Andruniów et al 2004) in general agreement with the results of this work and previous Raman studies (Kukura et al 2005;Yan et al 2004). In addition, two earlier reports (Gascon et al 2006;Bifone et al 1997) predict a torsional twist in the H-C11=C12-H moiety of approximately 20°, but subsequent ones (Frutos et al 2007;Polli et al 2010;Schapiro et al 2011;Andruniów et al 2004) all converge upon a twist of 35°-40°.…”

“…Nevertheless, we were able to use a deconvolution technique to extract information on the H-C11-C12-H torsional angle, establishing a deviation from planarity of at least 40°in bathorhodopsin. These findings agree with previous Raman observations (Kukura et al 2005;Yan et al 2004). This strong geometric distortion implies that a significant part of the light energy, used to drive the subsequent protein conformational changes that activate rhodopsin, is stored as steric strain in the bathorhodopsin retinylidene chain.…”

“…The mechanism for storing this large amount of energy, which corresponds to about 134 ± 12 kJ mol -1 (32 ± 3 kcal/mol), is not yet completely known. Three different mechanisms have been proposed and widely discussed (Honig et al 1979;Kukura et al 2005;Yan et al 2004;Birge et al 1998;Nishimura et al 1997a, b;Kandori and Maeda 1995;Ganter et al 1988;Rath et al 1998;Gascon et al 2006;Schreiber et al 2006;Rohrig et al 2004;Schreiber and Buss 2003): electrostatic energy storage by charge separation between the protonated Schiff base and a counterion (most likely Glu113 or a more extended complex of ions); mechanical energy storage in the form of steric strain energy; and van der Waals interactions between the chromophore and the protein. The first model proposed in 1979 by Honig et al (1979) considered the charge separation between the negatively charged counterion and the positively charged PSB as the main mechanism.…”

“…The first model proposed in 1979 by Honig et al (1979) considered the charge separation between the negatively charged counterion and the positively charged PSB as the main mechanism. Later Raman studies, however, suggested that intramolecular strain also gives a major contribution to the energy storage (Kukura et al 2005;Yan et al 2004). According to calculations by Birge et al (Birge et al 1998) about the 60 % of the stored energy can be attributed to strain and the rest of it to electrostatic effects.…”

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

“…As noted above, the conformational strain is manifested by HOOP modes in resonance Raman and Fourier transform infrared spectroscopy 56,62 . Following 11-cis to trans isomerization, a gradual reduction in HOOP mode intensity of the retinylidene chromophore is seen in the various rhodopsin photointermediates 56,64,77 . A similar progressive diminution is evident in the visible CD of the rhodopsin intermediates 55 , which agrees with the 2 H NMR analysis.…”