Enantioselective binding of an 11-cis-locked cyclopropyl retinal. The conformation of retinal in bovine rhodopsin

[formula: see text] The conformation of the retinal chromophore in rhodopsin is central for understanding the visual transduction process. The absolute twist around the 12-s bond of the chromophore in rhodopsin has been determined by studies with 11-cis-locked 11,12-cyclopropylretinal analogues (11S,12R)-2 and (11R,12S)-3, enantioselectively synthesized with the aid of an enzyme. The finding that enantiomer 2 binds to opsin while the other 3 does not defines the absolute sense of twist around the 12-s bond.     

The opsin shift and mechanism of spectral tuning in rhodopsin

Molecular dynamics simulations and combined quantum mechanical and molecular mechanical calculations have been performed to investigate the mechanism of the opsin shift and spectral tuning in rhodopsin. A red shift of -980 cm(-1) was estimated in the transfer of the chromophore from methanol solution environment to the protonated Schiff base (PSB)-binding site of the opsin. The conformational change from a 6-s-cis-all-trans configuration in solution to the 6-s-cis-11-cis conformer contributes additional -200 cm(-1), and the remaining effects were attributed to dispersion interactions with the aromatic residues in the binding site. An opsin shift of 2100 cm(-1) was obtained, in reasonable accord with experiment (2730 cm(-1)). Dynamics simulations revealed that the 6-s-cis bond can occupy two main conformations for the β-ionone ring, resulting in a weighted average dihedral angle of about -50°, which may be compared with the experimental estimate of -28° from solid-state NMR and Raman data. We investigated a series of four single mutations, including E113D, A292S, T118A, and A269T, which are located near the PSB, along the polyene chain of retinal and close to the ionone ring. The computational results on absorption energy shift provided insights into the mechanism of spectral tuning, which involves all means of electronic structural effects, including the stabilization or destabilization of either the ground or the electronically excited state of the retinal PSB.     

Photoaffinity labeling of bovine rhodopsin

Photoaffinity labeled (3-diazoacetoxy)-9-cis-retinal (1) and (9-methylenediazoacetoxy)-9-cis-retinal (20) were synthesized and bound to absorption maxima at 465 and 460 nm respectively. Binding studies established that synthetic retinals 1 and 2 bind to the natural binding site and that the integrity of the diazoacetoxy photoaffinity label is preserved in the process. Incorporation of 3-(O¹⁴COCHN₂)-labeled 9-cis retinal could be conveniently carried out in high yield using apomembrane solubilized in CHAPS as detergent to afford the pigment analog in a pure form. Photolysis of the diazoacetoxy group within the binding site led to 15–20%, crosslinking of rhodopsin as estimated by using radiocarbon containing labeled retinal 1 thus showing that this synthetic retinal is suitable for photoaffinity labeling of the active site in rhodopsin. Subsequent experiments to establish the site(s) of crosslinking by sequencing studies will then contribute to our knowledge of the structure of rhodopsin.     

An opsin shift in rhodopsin: retinal S0-S1 excitation in protein, in solution, and in the gas phase

We considered a series of model systems for treating the photoabsorption of the 11-cis retinal chromophore in the protonated Schiff-base form in vacuum, solutions, and the protein environment. A high computational level, including the quantum mechanical-molecular mechanical (QM/MM) approach for solution and protein was utilized in simulations. The S0-S1 excitation energies in quantum subsystems were evaluated by means of an augmented version of the multiconfigurational quasidegenerate perturbation theory (aug-MCQDPT2) with the ground-state geometry parameters optimized in the density functional theory PBE0/cc-pVDZ approximation. The computed positions of absorption bands lambdamax, 599(g), 448(s), and 515(p) nm for the gas phase, solution, and protein, respectively, are in excellent agreement with the corresponding experimental data, 610(g), 445(s), and 500(p) nm. Such consistency provides a support for the formulated qualitative conclusions on the role of the chromophore geometry, environmental electrostatic field, and the counterion in different media. An essentially nonplanar geometry conformation of the chromophore group in the region of the C14-C15 bond was obtained for the protein, in particular, owing to the presence of the neighboring charged amino acid residue Glu181. Nonplanarity of the C14-C15 bond region along with the influence of the negatively charged counterions Glu181 and Glu113 are found to be important to reproduce the spectroscopic features of retinal chromophore inside the Rh cavity. Furthermore, the protein field is responsible for the largest bond-order decrease at the C11-C12 double bond upon excitation, which may be the reason for the 11-cis photoisomerization specificity.     

The amino- and carboxyl-terminal sequence of bovine rhodopsin

The amino terminus of bovine rhodopsin is blocked and has the sequence x-Met-Asn(CHO)-Gly-Thr-Glu-Gly-Pro-Asn-Phe-Tyr-Val-Pro-Phe-Ser-Asn(CHO)-Lys-Thr-Gly-Val-Val-Arg, where CHO represents sites of carbohydrate attachment. The carboxyl-terminal sequence of rhodopsin is Val-Ser-Lys-Thr-Glu-Thr-Ser-Gln-Val-Ala-Pro-Ala. Upon short-term digestion of rod outer segment (ROS) membranes with thermolysin, opsin (similar to 35,000 daltons) is converted to a membrane-bound fragment O' (similar to 30,500 daltons) and 2 peptides containing 12 amino acids are released from the carboxyl terminus of rhodopsin into the supernatant. Upon long-term digestion of ROS with thermolysin, opsin and O' are replaced by the membrane-bound fragments F1 (similar to 25,000 daltons), and F2 (similar 9,500 daltons). When 32P-ROS are digested, F2 carries the 32P. Both O' and F1 contain the amino-terminal glycopeptide.     

Vibrational polarization and opsin shift of retinal schiff bases: theoretical study

The changes in the electronic excitation energy arising from molecular structural displacement induced by external electric field (so-called vibrational polarization) are examined theoretically for the protonated and neutral 11-cis retinal Schiff bases. It is shown that the magnitude of the field-induced structural displacement is significantly large for the protonated species, so that the change in the electronic excitation energy arising from this structural displacement is of the same order of magnitude as that arising from the direct effect of electric field on the electronic wave function. These two effects contribute additively to the field-induced spectral shift. The intensity-carrying mode (ICM) theory is employed to extract a single vibrational mode (called primary infrared ICM) that is most important for the field-induced structural displacement. A simple one-dimensional model is constructed, and the extent to which we can interpret the field-induced spectral shift by such a model is examined. In the case of the neutral species, only a small change in the electronic excitation energy is induced by external electric field, mainly because the vibrational polarizability of this species is small. The meaning of these results in the spectral tuning of visual pigments is discussed.     

Quantum chemical modeling of rhodopsin mutants displaying switchable colors

We look at the possibility to compute and understand the color change occurring upon mutation of a photochromic protein. Accordingly, ab initio multiconfigurational quantum chemical methods are used to construct basic quantum-mechanics/molecular-mechanics (QM/MM) models for a small mutant library of the sensory rhodopsin of Anabaena (Nostoc) sp. PCC7120 cyanobacterium. Together with the wild-type forms, a set of 26 absorption maxima spanning a ca. 80 nm range is obtained. We show that these models can be used to capture the electrostatic change controlling the computed color variation and the change in the ionization of specific side chains.     

7-cis-porphyropsin from 7-cis-3-dehydroretinal and cattle opsin.

Abstract 7-cis-3-Dehydroretinol, prepared by photoisomerization of the all-trans isomer in a polar solvent, was found to combine with cattle opsin to form a visual pigment analogue with an absorption maximum at 464 nm. The pigment is only moderately stable in hydroxylamine or in Ammonyx LO. and cannot be purified by column chromatography using Ammonyx LO. 9-cis-Porphyropsin, prepared in a manner similar to that reported by Azuma et al., is stable and has been purified by passing through a hydroxylapatite column. Three fractions were collected with eluant of increasing ionic strength. All fractions exhibit similar absorption properties with Λ_max at 498 nm. These results further indicate that the binding of opsin is a flexible one.     

Depsipeptides Communication 31. Synthesis of depsipeptides containing α-hydroxy α-amino acid residues

Summary Depsipeptides containing -hydroxy -amino acid residues as hydroxy-acid component were synthesized.Communication 11 of the series -Substituted -Amino Acids.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 11, pp. 1987–1992, November 1965     

Synthesis of o-glycosides of dipeptides containingβ-hydroxy amino acids

Conclusions The O--D-galactopyranosides of some serine-containing dipeptides were synthesized.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 9, pp. 2092–2094, September, 1971.     

Photochemistry of a retinal protonated schiff-base analogue mimicking the opsin shift of bacteriorhodopsin

A retinal Schiff base analogue which artificially mimics the protein-induced red shifting of absorption in bacteriorhodopsin (BR) has been investigated with femtosecond multichannel pump probe spectroscopy. The objective is to determine if the catalysis of retinal internal conversion in the native protein BR, which absorbs at 570 nm, is directly correlated with the protein-induced Stokes shifting of this absorption band otherwise known as the "opsin shift". Results demonstrate that the red shift afforded in the model system does not hasten internal conversion relative to that taking place in a free retinal-protonated Schiff base (RPSB) in methanol solution, and stimulated emission takes place with biexponential kinetics and characteristic timescales of approximately 2 and 10.5 ps. This shows that interactions between the prosthetic group and the protein that lead to the opsin shift in BR are not directly involved in reducing the excited-state lifetime by nearly an order of magnitude. A sub-picosecond phase of spectral evolution, analogues of which are detected in photoexcited retinal proteins and RPSBs in solution, is observed after excitation anywhere within the intense visible absorption band. It consists of a large and discontinuous spectral shift in excited-state absorption and is assigned to electronic relaxation between excited states, a scenario which might also be relevant to those systems as well. Finally, a transient excess bleach component that tunes with the excitation wavelength is detected in the data and tentatively assigned to inhomogeneous broadening in the ground state absorption band. Possible sources of such inhomogeneity and its relevance to native RPSB photochemistry are discussed.     

Quantum yield of Ce3+ and energy transfer between Ce3+ and Tb3+ in borax glasses

Quantum yields of Ce³⁺ in borax glasses were obtained by the comparative method and by lifetime measurements. Energy transfer from Ce³⁺ to Tb³⁺ was detected in borax glasses from the excitation spectrum. The transfer probabilities were calculated from the increase in the Tb³⁺ fluorescence in the presence of Ce³⁺ and the decrease of the Ce³⁺ fluorescence in the presence of Tb³⁺. A linear dependence of the transfer probabilities was found with the squared sum of the concentrations of the donor and acceptor ions. This is consistent with dipolar mechanism and interactions of one Ce³⁺ donor with two Tb³⁺ acceptors, in view of the Fong-Diestler theory.     

Effect of polarization on the opsin shift in rhodopsins. 1. A combined QM/QM/MM model for bacteriorhodopsin and pharaonis sensory rhodopsin II

The optical and IR-spectroscopic properties of the protonated Schiff base of retinal are highly sensitive to the electrostatic environment. This feature makes retinal a useful probe to study structural differences and changes in rhodopsins. It also raises an interest to theoretically predict the spectroscopic response to mutation and structural evolution. Computational models appropriate for this purpose usually combine sophisticated quantum mechanical (QM) methods with molecular mechanics (MM) force fields. In an effort to test and improve the accuracy of these QM/MM models, we consider in this article the effects of polarization and inter-residual charge transfer within the binding pocket of bacteriorhodopsin (bR) and pharaonis sensory rhodopsin II (psRII, also called pharaonis phoborhodopsin, ppR) on the excitation energy using an ab initio QM/QM/MM approach. The results will serve as reference for assessing empirical polarization models in a consecutive article.     

Mechanical properties of bovine rhodopsin and bacteriorhodopsin: possible roles in folding and function

Molecular interactions and mechanical properties that contribute to the stability and function of proteins are complex and of fundamental importance. In this study, we used single-molecule dynamic force spectroscopy (DFS) to explore the interactions and the unfolding energy landscape of bovine rhodopsin and bacteriorhodopsin. An analysis of the experimental data enabled the extraction of parameters that provided insights into the kinetic stability and mechanical properties of these membrane proteins. Individual structural segments of rhodopsin and bacteriorhodopsin have different properties. A core of rigid structural segments was observed in rhodopsin but not in bacteriorhodopsin. This core may reflect differences in mechanisms of protein folding between the two membrane proteins. The different structural rigidity of the two proteins may also reflect their adaptation to differing functions.     

Assigning the resonance Raman spectral features of rhodopsin, isorhodopsin and bathorhodopsin in bovine photostationary state spectra.

Abstract—High resolution resonance Raman spectra of rhodopsin. isorhodopsin and photostationary state mixtures containing a high percentage of bathorhodopsin arc presented. New spectral features are detected which were not obsei-ved in lower resolution studies by other workers. All of the hands in the photostationary state spcctra arc assigned based on pure rhodopsin and isorhodopsin resonance Raman results and alterations in the photostationary state mixture. The spectral features in these spectra are invariant from 20 to 150K indicating that retinal and protein structural alteration, consistent with a model of excitation proposed by Lewis, occurs in steady-state spectra even at 20 K. In addition, the relative intensity of certain features in the photostationary state spectra are altered upon D₂O suspension. One explanation for these alterations is that the contributions of various intcrmediates to the photostationary state mixture are changed when membrane fragments are suspended in D₂O.     

UV laser photochemistry and diborane at 193 nm: Quantum yield for BH3 production

The quantum yield ø_BH3 for BH₃ production from ArF-laser-excited B₂H₆ was determined to be 2.00 ± 0.25 by trapping with PF₃. The former postulate of BH₃ as a chain carrier in ArF-laser-induced B₂H₆-D₂ exchange is confirmed.     

Quantum yield spectra of vanadium in tetrahedral [VO4]3− complexes

Conclusions Studies of VL_2,3 quantum yield spectra and analysis of theoretical and experimental data were used to give information on the processes proceeding during the emission and absorption of the X-ray VL-radiation. It is very probable that the X-ray L₃-absorption processes in compounds where the 3d-metal has the highest valence proceed in the presence of an additional electron on one of the vacant orbitals (for tetrahedral complexes on the 2e-MO). This assumption can confirm the hypothesis on the reemissional transitions and explain the energy divergence between the VL-emission and absorption spectra.Institute of Physics of Metals, Ural Branch Academy Sciences of the USSR. Translated from Zhurnal Strukturnoi Khimii, Vol. 29, No. 1, pp. 81–86, January–February, 1988.     

Modulating rhodopsin receptor activation by altering the pKa of the retinal Schiff base

The visual pigment rhodopsin is a seven-transmembrane (7-TM) G protein-coupled receptor (GPCR). Activation of rhodopsin involves two pH-dependent steps: proton uptake at a conserved cytoplasmic motif between TM helices 3 and 6, and disruption of a salt bridge between a protonated Schiff base (PSB) and its carboxylate counterion in the transmembrane core of the receptor. Formation of an artificial pigment with a retinal chromophore fluorinated at C14 decreases the intrinsic pKa of the PSB and thereby destabilizes this salt bridge. Using Fourier transform infrared difference and UV-visible spectroscopy, we characterized the pH-dependent equilibrium between the active photoproduct Meta II and its inactive precursor, Meta I, in the 14-fluoro (14-F) analogue pigment. The 14-F chromophore decreases the enthalpy change of the Meta I-to-Meta II transition and shifts the Meta I/Meta II equilibrium toward Meta II. Combining C14 fluorination with deletion of the retinal beta-ionone ring to form a 14-F acyclic artificial pigment uncouples disruption of the Schiff base salt bridge from transition to Meta II and in particular from the cytoplasmic proton uptake reaction, as confirmed by combining the 14-F acyclic chromophore with the E134Q mutant. The 14-F acyclic analogue formed a stable Meta I state with a deprotonated Schiff base and an at least partially protonated protein counterion. The combination of retinal modification and site-directed mutagenesis reveals that disruption of the protonated Schiff base salt bridge is the most important step thermodynamically in the transition from Meta I to Meta II. This finding is particularly important since deprotonation of the retinal PSB is known to precede the transition to the active state in rhodopsin activation and is consistent with models of agonist-dependent activation of other GPCRs.