Constraints of opsin structure on the ligand-binding site: studies with ring-fused retinals

Ring-fused retinal analogs were designed to examine the hula-twist mode of the photoisomerization of the 9-cis retinylidene chromophore. Two 9-cis retinal analogs, the C11-C13 five-membered ring-fused and the C12-C14 five-membered ring-fused retinal derivatives, formed the pigments with opsin. The C11-C13 ring-fused analog was isomerized to a relaxed all-trans chromophore (lambda(max) > 400 nm) at even -269 degrees C and the Schiff base was kept protonated at 0 degrees C. The C12-C14 ring-fused analog was converted photochemically to a bathorhodopsin-like chromophore (lambda(max) = 583 nm) at -196 degrees C, which was further converted to the deprotonated Schiff base at 0 degrees C. The model-building study suggested that the analogs do not form pigments in the retinal-binding site of rhodopsin but form pigments with opsin structures, which have larger binding space generated by the movement of transmembrane helices. The molecular dynamics simulation of the isomerization of the analog chromophores provided a twisted C11-C12 double bond for the C12-C14 ring-fused analog and all relaxed double bonds with a highly twisted C10-C11 bond for the C11-C13 ring-fused analog. The structural model of the C11-C13 ring-fused analog chromophore showed a characteristic flip of the cyclohexenyl moiety toward transmembrane segments 3 and 4. The structural models suggested that hula twist is a primary process for the photoisomerization of the analog chromophores.     

NMR chemical shifts of the rhodopsin chromophore in the dark state and in bathorhodopsin: a hybrid QM/MM molecular dynamics study

We investigate nuclear magnetic resonance (NMR) parameters of the rhodopsin chromophore in the dark state of the protein and in the early photointermediate bathorhodopsin via first-principles molecular dynamics simulations and NMR chemical shift calculations in a hybrid quantum/classical (QM/MM) framework. NMR parameters are particularly sensitive to structural properties and to the chemical environment, which allows us to address different questions about the retinal chromophore in situ. Our calculations show that both the 13C and the 1H NMR chemical shifts are rather insensitive to the protonation state of Glu181, an ionizable amino acid side chain located in the vicinity of the isomerizing 11-cis bond. Thus, other techniques should be better suited to establish its protonation state. The calculated chemical shifts for bathorhodopsin further support our previously published theoretical structure, which is in very good agreement with more recent X-ray data.     

The role of the beta-ionone ring in the photochemical reaction of rhodopsin

Time-dependent density functional theory (TDDFT) calculations on the photoabsorption process of the 11-cis retinal protonated Schiff base (PSB) chromophore show that the Franck-Condon relaxation of the first excited state of the chromophore involves a torsional twist motion of the beta-ionone ring relative to the conjugated retinyl chain. For the ground state, the beta-ionone ring and the retinyl chain of the free retinal PSB chromophore form a -40 degrees dihedral angle as compared to -94 degrees for the first excited state. The double bonds of the retinal are shorter for the fully optimized structure of the excited state than for the ground state suggesting a higher cis-trans isomerization barrier for the excited state than for the ground state. According to the present TDDFT calculations, the excitation of the retinal PSB chromophore does not primarily lead to a reaction along the cis-trans torsional coordinate at the C11-C12 bond. The activation of the isomerization center seems to occur at a later stage of the photo reaction. The results obtained at the TDDFT level are supported by second-order M?ller-Plesset (MP2) and approximate singles and doubles-coupled cluster (CC2) calculations on retinal chromophore models; the MP2 and CC2 calculations yield for them qualitatively the same ground state and excited-state structures as obtained in the density functional theory and TDDFT calculations.     

A semiempirical study of the optimized ground and excited state potential energy surfaces of retinal and its protonated Schiff base.

The electronic ground and first excited states of retinal and its Schiff base are optimized for the first time using the semiempirical AM1 Hamiltonian. The barrier for rotation about the C(11)-C(12) double bond is characterized by variation of both the twist angle delta(C(10)-C(11)-C(12)-C(13)) and the bond length d(C(11)-C(12)). The potential energy surface is obtained by varying these two parameters. The calculated ground state rotational barrier is equal to 15.6 kcal/mol for retinal and 20.5 kcal/mol for its Schiff base. The all-trans conformation is more stable by 3.7 kcal/mol than the 11-cis geometry. For the first excited state, S(1,) the 90 degrees twisted geometry represents a saddle point for retinal with the rotational barrier of 14.6 kcal/mol. In contrast, this conformation is an energy minimum for the Schiff base. It can be easily reached at room temperature from the planar minima since it is separated from them by a barrier of only 0.6 kcal/mol. The 90 degrees minimum conformation is more stable than the all-trans by 8.6 kcal/mol. We are thus able to present a reaction path on the S(1) surface of the retinal Schiff base with an almost barrier-less geometrical relaxation into a twisted minimum geometry, as observed experimentally. The character of the ground and first excited singlet states underscores the need for the inclusion of double excitations in the calculations.     

Excitation energies of retinal chromophores: critical role of the structural model

We employ a variety of highly-correlated approaches including quantum Monte Carlo (QMC) and the n-electron valence state perturbation theory (NEVPT2) to compute the vertical excitation energies of retinal protonated Schiff base (RPSB) models in the gas phase. We find that the NEVPT2 excitation energies are in good agreement with the QMC values and confirm our previous findings that the complete-active-space perturbation (CASPT2) approach yields accurate excitations for RPSB models only when the more recent zero-order IPEA Hamiltonian is employed. The excitations computed with the original zero-order formulation of CASPT2 are instead systematically red-shifted by more than 0.3 eV. We then focus on the full 11-cis retinal chromophore and show that the M06-2X and MP2 approaches provide reliable ground-state equilibrium structures for this system while the complete-active-space self-consistent field (CASSCF) geometry is characterized by significantly higher ground-state energies at the NEVPT2 and CASPT2 level. Our calibration of the structural model together with the general agreement of all highly-correlated excited-state methods allows us to reliably assign a value of about 2.3 eV to the vertical excitation of 11-cis RPSB in the gas-phase.     

Primary events in dim light vision: a chemical and spectroscopic approach toward understanding protein/chromophore interactions in rhodopsin

The visual pigment rhodopsin (bovine) is a 40 kDa protein consisting of 348 amino acids, and is a prototypical member of the subfamily A of G protein-coupled receptors (GPCRs). This remarkably efficient light-activated protein (quantum yield = 0.67) binds the chromophore 11-cis-retinal covalently by attachment to Lys296 through a protonated Schiff base. The 11-cis geometry of the retinylidene chromophore keeps the partially active opsin protein locked in its inactive state (inverse agonist). Several retinal analogs with defined configurations and stereochemistry have been incorporated into the apoprotein to give rhodopsin analogs. These incorporation results along with the spectroscopic properties of the rhodopsin analogs clarify the mode of entry of the chromophore into the apoprotein and the biologically relevant conformation of the chromophore in the rhodopsin binding site. In addition, difference UV, CD, and photoaffinity labeling studies with a 3-diazo-4-oxo analog of 11-cis-retinal have been used to chart the movement of the retinylidene chromophore through the various intermediate stages of visual transduction.     

(9Z)- and (11Z)-8-methylretinals for artificial visual pigment studies: stereoselective synthesis, structure, and binding models

Artificial visual pigment formation was studied by using 8-methyl-substituted retinals in an effort to understand the effect that alkyl substitution of the chromophore side chain has on the visual cycle. The stereoselective synthesis of the 9-cis and 11-cis isomers of 8-methylretinal, as well as the 5-demethylated analogues is also described. The key bond formations consist of a thallium-accelerated Suzuki cross-coupling reaction between cyclohexenylboronic acids and dienyliodides (C6-C7), and a highly stereocontrolled Horner-Wadsworth-Emmons or Wittig condensation (C11-C12). The cyclohexenylboronic acid was prepared by trapping the precursor cyclohexenyllithium species with B(OiPr)(3) or B(OMe)(3). The cyclohexenyllithium species is itself obtained by nBuLi-induced elimination of a trisylhydrazone (Shapiro reaction), or depending upon the steric hindrance of the ring, by iodine-metal exchange. In binding experiments with the apoprotein opsin, only 9-cis-5-demethyl-8-methylretinal yielded an artificial pigment; 9-cis-8-methylretinal simply provided residual binding, while evidence of artificial pigment formation was not found for the 11-cis analogues. Molecular-mechanics-based docking simulations with the crystal structure of rhodopsin have allowed us to rationalize the lack of binding displayed by the 11-cis analogues. Our results indicate that these isomers are highly strained, especially when bound, due to steric clashes with the receptor, and that these interactions are undoubtedly alleviated when 9-cis-5-demethyl-8-methylretinal binds opsin.     

Interaction of chromophore, 11-<Emphasis Type="Italic">cis</Emphasis>-retinal,with amino acid residues of the visual pigment rhodopsin in the region of protonated Schiff base: A molecular dynamics study

A molecular dynamics study of the dark adapted visual pigment rhodopsin molecule was carried out. The interaction of the chromophore group, 11-cis-retinal, with the nearest amino acid residues in the chromophore center of the molecule, namely, in the region of the protonated Schiff base linkage, was analyzed. Most likely, the interaction of the CH=NH bond with the negatively charged amino acid residue Glu113 cannot be described as a simple electrostatic interaction of two oppositely charged groups. One can propose that not only Glu113 but also Glu181 and Ser186 are involved in stabilization of the protonated Schiff base linkage. Accord-ing to calculations, Glu181 interacts, as the counter-ion, with the Schiff base indirectly via Ser186. The intramolecular mechanisms of protonated Schiff base stabilization in rhodopsin are discussed. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 1, pp. 19–27, January, 2007.     

Torsion potential works in rhodopsin

We investigate the role of protein environment of rhodopsin and the intramolecular interaction of the chromophore in the cis-trans photoisomerization of rhodopsin by means of a newly developed theoretical method. We theoretically produce modified rhodopsins in which a force field of arbitrarily chosen part of the chromophore or the binding pocket of rhodopsin is altered. We compare the equilibrium conformation of the chromophore and the energy stored in the chromophore of modified rhodopsins with those of native rhodopsins. This method is called site-specific force field switch (SFS). We show that this method is most successfully applied to the torsion potential of rhodopsin. Namely, by reducing the twisting force constant of the C11=C12 of 11-cis retinal chromophore of rhodopsin to zero, we found that the equilibrium value of the twisting angle of the C11=C12 bond is twisted in the negative direction down to about -80 degrees. The relaxation energy obtained by this change amounts to an order of 10 kcal/mol. In the case that the twisting force constant of the other double bond is reduced to zero, no such large twisting of the bond happens. From these results we conclude that a certain torsion potential is applied specifically to the C11=C12 bond of the chromophore in the ground state of rhodopsin. This torsion potential facilitates the bond-specific cis-trans photoisomerization of rhodopsin. This kind of the mechanism is consistent with our torsion model proposed by us more than a quarter of century ago. The origin of the torsion potential is analyzed in detail on the basis of the chromophore structure and protein conformation, by applying the SFS method extensively.     

Stairway to the conical intersection: a computational study of the retinal isomerization

The potential-energy surface of the first excited state of the 11-cis-retinal protonated Schiff base (PSB11) chromophore has been studied at the density functional theory (DFT) level using the time-dependent perturbation theory approach (TDDFT) in combination with Becke's three-parameter hybrid functional (B3LYP). The potential-energy curves for torsion motions around single and double bonds of the first excited state have also been studied at the coupled-cluster approximate singles and doubles (CC2) level. The corresponding potential-energy curves for the ground state have been calculated at the B3LYP DFT and second-order M?ller-Plesset (MP2) levels. The TDDFT study suggests that the electronic excitation initiates a turn of the beta-ionone ring around the C6-C7 bond. The torsion is propagating along the retinyl chain toward the cis to trans isomerization center at the C11=C12 double bond. The torsion twist of the C10-C11 single bond leads to a significant reduction in the deexcitation energy indicating that a conical intersection is being reached by an almost barrierless rotation around the C10-C11 single bond. The energy released when passing the conical intersection can assist the subsequent cis to trans isomerization of the C11=C12 double bond. The CC2 calculations also show that the torsion barrier for the twist of the retinyl C10-C11 single bond adjacent to the isomerization center almost vanishes for the excited state. Because of the reduced torsion barriers of the single bonds, the retinyl chain can easily deform in the excited state. Thus, the CC2 and TDDFT calculations suggest similar reaction pathways on the potential-energy surface of the excited state leading toward the conical intersection and resulting in a cis to trans isomerization of the retinal chromophore. According to the CC2 calculations the cis to trans isomerization mechanism does not involve any significant torsion motion of the beta-ionone ring.     

Recent bioorganic studies on rhodopsin and visual transduction

Rhodopsin, the pigment responsible for vision in animals, insect and fish is a typical G protein (guanyl-nucleotide binding protein) consisting of seven transmembrane alpha helices and their interconnecting extramembrane loops. In the case of bovine rhodopsin, the best studied of the visual pigments, the chromophore is 11-cis retinal attached to the terminal amino group of Lys296 through a protonated Schiff base linkage. Photoaffinity labeling with a 3-diazo-4-oxo-retinoid shows that C-3 of the ionone ring moiety is close to Trp265 in helix F (VI) in dark inactivated rhodopsin. Irradiation causes a cis to trans isomerization of the 11-cis double bond giving rise to the highly strained intermediate bathorhodopsin. This undergoes a series of thermal relaxation through lumi-, meta-I and meta-II intermediates after which the retinal chromophore is expelled from the opsin binding pocket. Photoaffinity labeling performed with 3-diazo-4-oxoretinal at -196 degrees C for batho-, -80 degrees C for lumi-, -40 degrees C for meta-I, and 0 degrees C for meta-II rhodopsin showed that in bathorhodopsin the ring is still close to Trp265. However, in lumi-, meta-I and meta-II intermediates crosslinking occurs unexpectedly at A169 in helix D (IV). This shows that large movements in the helical arrangements and a flip over of the ring moiety accompanies the transduction (or bleaching) process. These changes in retinal/opsin interactions are necessarily accompanied by movements of the extramembrane loops, which in turn lead to activation of the G protein residing in the cytoplasmic side. Of the numerous G protein coupled receptors, this is the first time that the outline of transduction pathway has been clarified.     

Glutamic acid 181 is negatively charged in the bathorhodopsin photointermediate of visual rhodopsin

Assignment of the protonation state of the residue Glu-181 is important to our understanding of the primary event, activation processes and wavelength selection in rhodopsin. Despite extensive study, there is no general agreement on the protonation state of this residue in the literature. Electronic assignment is complicated by the location of Glu-181 near the nodal point in the electrostatic charge shift that accompanies excitation of the chromophore into the low-lying, strongly allowed ππ* state. Thus, the charge on this residue is effectively hidden from electronic spectroscopy. This situation is resolved in bathorhodopsin, because photoisomerization of the chromophore places Glu-181 well within the region of negative charge shift following excitation. We demonstrate that Glu-181 is negatively charged in bathorhodopsin on the basis of the shift in the batho absorption maxima at 10 K [λ(max) band (native) = 544 ± 2 nm, λ(max) band (E181Q) = 556 ± 3 nm] and the decrease in the λ(max) band oscillator strength (0.069 ± 0.004) of E181Q relative to that of the native protein. Because the primary event in rhodopsin does not include a proton translocation or disruption of the hydrogen-bonding network within the binding pocket, we may conclude that the Glu-181 residue in rhodopsin is also charged.     

DEPENDENCE OF THE TRIPLET YIELD ON EXCITATION ENERGY IN ALL-TRANS AND 11-CIS RETINAL*

Abstract. The triplet-triplet absorption of all-trans and 11- cis retinal was measured as a function of the exciting radiation from 423 nm to 365 nm in a glass of 3-methylpentane at 77 K. This experiment was also accomplished with all-trans retinal in hexane at ambient temperature. The relative triplet formation quantum yields of all-trans and 11-cis retinal at 77 K were found to be independent (±10%) of the frequency of the exciting radiation. At room temperature we measured an increase in this relative quantum yield for all- trans retinal from 1.0 at 365 nm to 1.82 at 423 nm [Bensasson et al. (1975) measured an absolute quantum yield of 0.45 at 353 nm]. These results are used to evaluate previous interpretations for photophysical decay processes in all-trans retinal, and previous suggestions for wavelength dependent radiationless transitions are shown to be unacceptable. High energy excitation of 300 K solutions of all- trans retinal produce excited states that result in less efficient intersystem crossing. These states appear to be inaccessible in the 77 K matrices. We suggest that steric restrictions introduced by the retinal matrix interaction at 77 K are able to block this new internal conversion pathway back to the ground state.     

Retinal counterion switch mechanism in vision evaluated by molecular simulations

Photoisomerization of the retinylidene chromophore of rhodopsin is the starting point in the vision cascade. A counterion switch mechanism that stabilizes the retinal protonated Schiff base (PSB) has been proposed to be an essential step in rhodopsin activation. On the basis of vibrational and UV-visible spectroscopy, two counterion switch models have emerged. In the first model, the PSB is stabilized by Glu181 in the meta I state, while in the most recent proposal, it is stabilized by Glu113 as well as Glu181. We assess these models by conducting a pair of microsecond scale, all-atom molecular dynamics simulations of rhodopsin embedded in a 99-lipid bilayer of SDPC, SDPE, and cholesterol (2:2:1 ratio) varying the starting protonation state of Glu181. Theoretical simulations gave different orientations of retinal for the two counterion switch mechanisms, which were used to simulate experimental 2H NMR spectra for the C5, C9, and C13 methyl groups. Comparison of the simulated 2H NMR spectra with experimental data supports the complex-counterion mechanism. Hence, our results indicate that Glu113 and Glu181 stabilize the retinal PSB in the meta I state prior to activation of rhodopsin.     

Calculating absorption shifts for retinal proteins: computational challenges

Rhodopsins can modulate the optical properties of their chromophores over a wide range of wavelengths. The mechanism for this spectral tuning is based on the response of the retinal chromophore to external stress and the interaction with the charged, polar, and polarizable amino acids of the protein environment and is connected to its large change in dipole moment upon excitation, its large electronic polarizability, and its structural flexibility. In this work, we investigate the accuracy of computational approaches for modeling changes in absorption energies with respect to changes in geometry and applied external electric fields. We illustrate the high sensitivity of absorption energies on the ground-state structure of retinal, which varies significantly with the computational method used for geometry optimization. The response to external fields, in particular to point charges which model the protein environment in combined quantum mechanical/molecular mechanical (QM/MM) applications, is a crucial feature, which is not properly represented by previously used methods, such as time-dependent density functional theory (TDDFT), complete active space self-consistent field (CASSCF), and Hartree-Fock (HF) or semiempirical configuration interaction singles (CIS). This is discussed in detail for bacteriorhodopsin (bR), a protein which blue-shifts retinal gas-phase excitation energy by about 0.5 eV. As a result of this study, we propose a procedure which combines structure optimization or molecular dynamics simulation using DFT methods with a semiempirical or ab initio multireference configuration interaction treatment of the excitation energies. Using a conventional QM/MM point charge representation of the protein environment, we obtain an absorption energy for bR of 2.34 eV. This result is already close to the experimental value of 2.18 eV, even without considering the effects of protein polarization, differential dispersion, and conformational sampling.     

A molecular spring for vision

Light absorption by the visual pigment rhodopsin leads to vision via a complex signal transduction pathway that is initiated by the ultrafast and highly efficient photoreaction of its chromophore, the retinal protonated Schiff base (RPSB). Here, we investigate this reaction in real time by means of unrestrained molecular dynamics simulations of the protein in a membrane mimetic environment, treating the chromophore at the density functional theory level. We demonstrate that a highly strained all-trans RPSB is formed starting from the 11-cis configuration (dark state) within approximately 100 fs by a minor rearrangement of the nuclei under preservation of the saltbridge with Glu113 and virtually no deformation of the binding pocket. Hence, the initial step of vision can be understood as the compression of a molecular spring by a minor change of the nuclear coordinates. This spring can then release its strain by altering the protein environment.     

Spectral tuning in visual pigments: an ONIOM(QM:MM) study on bovine rhodopsin and its mutants

We have investigated geometries and excitation energies of bovine rhodopsin and some of its mutants by hybrid quantum mechanical/molecular mechanical (QM/MM) calculations in ONIOM scheme, employing B3LYP and BLYP density functionals as well as DFTB method for the QM part and AMBER force field for the MM part. QM/MM geometries of the protonated Schiff-base 11- cis-retinal with B3LYP and DFTB are very similar to each other. TD-B3LYP/MM excitation energy calculations reproduce the experimental absorption maximum of 500 nm in the presence of native rhodopsin environment and predict spectral shifts due to mutations within 10 nm, whereas TD-BLYP/MM excitation energies have red-shift error of at least 50 nm. In the wild-type rhodopsin, Glu113 shifts the first excitation energy to blue and accounts for most of the shift found. Other amino acids individually contribute to the first excitation energy but their net effect is small. The electronic polarization effect is essential for reproducing experimental bond length alternation along the polyene chain in protonated Schiff-base retinal, which correlates with the computed first excitation energy. It also corrects the excitation energies and spectral shifts in mutants, more effectively for deprotonated Schiff-base retinal than for the protonated form. The protonation state and conformation of mutated residues affect electronic spectrum significantly. The present QM/MM calculations estimate not only the experimental excitation energies but also the source of spectral shifts in mutants.     

Characterization and photochemistry of 13-desmethyl bacteriorhodopsin

The photochemistry of the 13-desmethyl (DM) analogue of bacteriorhodopsin (BR) is examined by using spectroscopy, molecular orbital theory, and chromophore extraction followed by conformational analysis. The removal of the 13-methyl group permits the direct photochemical formation of a thermally stable, photochemically reversible state, P1(DM) (lambda(max) = 525 nm), which can be generated efficiently by exciting the resting state, bR(DM) with yellow or red light (lambda > 590 nm). Chromophore extraction analysis reveals that the retinal configuration in P1(DM) is 9-cis, identical to that of the retinal configuration in the native BR P1 state. Fourier transform infrared and Raman experiments on P1(DM) indicate an anti configuration around the C15=N bond, as would be expected of an O-state photoproduct. However, low-temperature spectroscopy and ambient, time-resolved studies indicate that the P1(DM) state forms primarily via thermal relaxation from the L(D)(DM) state. Theoretical studies on the BR binding site show that 13-dm retinal is capable of isomerizing into a 9-cis configuration with minimal steric hindrance from surrounding residues, in contrast to the native chromophore in which surrounding residues significantly obstruct the corresponding motion. Analysis of the photokinetic experiments indicates that the Arrhenius activation energy of the bR(DM) --> P1(DM) transition in 13-dm-BR is less than 0.6 kcal/mol (vs 22 +/-5 kcal/mol measured for the bR --> P (P1 and P2) reaction in 85:15 glycerol:water suspensions of wild type). Consequently, the P1(DM) state in 13-dm-BR can form directly from all-trans, 15-anti intermediates (bR(DM) and O(DM)) or all-trans, 15-syn (K(D)(DM)/L(D)(DM)) intermediates. This study demonstrates that the 13-methyl group, and its interactions with nearby binding site residues, is primarily responsible for channeling one-photon photochemical and thermal reactions and is limited to the all-trans and 13-cis species interconversions in the native protein.     

EFFECT OF PROTONATION ON THE ISOMERIZATION PROPERTIES OF n-BUTYLAMINE SCHIFF BASE OF ISOMERIC RETINAL AS REVEALED BY DIRECT HPLC ANALYSES: SELECTION OF ISOMERIZATION PATHWAYS BY RETINAL PROTEINS

Alumina adsorption chromatography and ion-pair reversed-phase chromatography were developed to analyze the isomers of unprotonated and protonated n-butylamine Schiff base of retinal (RSB and PRSB), respectively. Photoisomerization starting from the all-trans, 11-cis and 13-cis isomers was traced for RSB in n-hexane, acetonitrile, methanol and 1-butanol, and for PRSB in methanol, acetonitrile and 1-butanol. The quantum yields of photoisomerization for the all-trans, 9-cis, 11-cis and 13-cis isomers were determined for RSB and PRSB in the above solvents except 1-butanol. On the other hand, photoisomerization of isomeric retinal bound (through Schiff base linkage) to bovine serum albumin (RBSA) in aqueous solution (pH 3, 7 and 12) as well as thermal isomerization of RSB (in n-hexane), PRSB (in methanol) and RBSA (in aqueous solution, pH 7) were traced starting from the all-trans, 11-cis, and 13-cis isomers. Protonation of RSB drastically changes the pathway of photoisomerization and increases the quantum yields of isomeric RSB. The solvent polarity increases the quantum yields of RSB differently depending on the configuration. Protonation enhances thermal isomerization also. The results of the above model systems are compared with those of retinal proteins to rationalize their selection of the particular isomerization pathways.