Retinal Isomerization Research Articles

The Effect of Protein Conformation Change from αII to αI on the Bacteriorhodopsin Photocycle

The bacteriorhodopsin (bR) photocycle was followed by use of time-resolved Fourier-transform infrared (FTIR) spectroscopy as a function of temperature (15–85°C) as the α II → α I conformational transition occurs. The photocycle rate increases with increasing temperature, but its efficiency is found to be drastically reduced as the transition takes place. A large shift is observed in the all- trans ↔ 13- cis equilibrium due to the increased stability of the 13- cis isomer in α I form. This, together with the increase in the rate of dark adaptation as the temperature increases, leads to a large increase in the 13- cis isomer concentration in bR in the α I form. The fact that 13- cis retinal has a much-reduced absorption cross-section and its inability to pump protons leads to an observed large reduction in the concentration of the observed photocycle intermediates, as well as the proton gradient at a given light intensity. These results suggest that nature might have selected the α II rather than the α I form as the helical conformation in bR to stabilize the all- trans retinal isomer that is a better light absorber and is capable of pumping protons.

Proton Polarizability of Hydrogen-Bonded Network and its Role in Proton Transfer in Bacteriorhodopsin

Room-temperature time-resolved step-scan Fourier Transform Infrared (FTIR) spectroscopy has been used to study the photocycle of native bacteriorhodopsin (bR) suspension in both H2O and D2O. The kinetics of the retinal isomerization, and that of the protonation/deprotonation of the proton acceptor, Asp85, are compared in the μs to ms time domain. It is found that hydrogen/deuterium (H/D) isotope exchange does not significantly affect the kinetics of the retinal isomerization and relaxation processes. However, the protonation/deprotonation processes of Asp85 COO- become slower in D2O. We also studied the kinetics of the continuum absorbance change in the 1850−1800 cm-1 frequency region, which has previously been proposed to correspond to the absorption of the delocalized proton that is involved in the proton transport to the surface during the photocycle. An H/D isotope shift of the frequency range of this continuum absorbance has been confirmed by the observation that the band in the 1850−1800 cm-1 disappears in the photocycle of bR in D2O. These results could support the previous proposal that the intramolecular proton release pathway consists of an H-bonded network. Our results also suggest that the two independent processes, the transfer of a proton from the Schiff base to Asp85 and the release of a different proton to the extracellular surface, are closely coupled events.

Molecular Biology of Light Transduction by the Mammalian Photoreceptor, Rhodopsin

Rhodopsin, the vertebrate photoreceptor, is a prototypic molecule in the largest family of G- protein coupled receptors (GPCR). Like all receptors of this family, it contains three distinct domains: the cytoplasmic (intracellular) domain that is involved in all the protein-protein interactions; the transmembrane (TM) domain where the signal transduction begins, by light- catalysed isomerization of 11-cis-retinal to all trans-retinal, and the intradiscal domain which appears to be involved in a specific tertiary structure. The main focus of this talk is to describe efforts to understand specific structure and function in each domain. The main findings to be presented are as follows: 1. Intradiscal domain contains a globular tertiary structure. A central feature is a disulfide bond (Cys110-Cys187) which is conserved in most of the known GPCR. 2. The correct folding in vivo requires the formation of the above disulfide bond. Misfolding resulting in non-retinal binding is frequently caused by Retinitis Pigmentosa (RP) point mutations in the intradiscal and the TM domain. 3. In vivo folding studies, using RP mutations in every one of the seven helices, have shown that the packing of the helices in the TM domain and folding to form the intradiscal tertiary structure are coupled. 4. Cysteine mutagenesis has been used systematically to study the tertiary structure and light-dependent changes throughout the cytoplasmic face by combination of biochemical and biophysical studies. In particular, EPR spectroscopy following spin labeling of selected double cysteine mutants has shown movements in helices, including tilting, following retinal isomerization. 5. Large scale expression of mutants has allowed application of both 19F-NMR (solution) and MAS solid state NMR (in collaboration with Dr. Steve Smith's group, SUNY, Stony Brook). Results of current work are promising for detailed study of the conformational change. Finally, a unifying hypothesis, which is termed the central dogma in the GPCR field, will be proposed. This states that despite the enormous variation in “accessory” structural details, the principal mechanism of signal transduction starting with pertubation in the seven helical bundle is fundamentally the same in all GPCRs. Experiments to test helix movements, the first step in signal transduction following ligand binding in two adrenergic receptors are now feasible. The patterns of helix movements in them will be compared with the pattern demonstrated for rhodopsin and its mutants.

Time-resolved thermodynamic changes photoinduced in 5,12-trans-locked bacteriorhodopsin. Evidence that retinal isomerization is required for protein activation.

Structural volume changes upon excitation of isomerization-blocked 5,12-trans-locked bacteriorhodopsin (bR) (bacterio-opsin + 5-12-trans-locked retinal) were studied using photothermal methods. The very small prompt expansion detected using laser-induced optoacoustics (0.3 mL/mol of absorbed photons) is assigned to a charge reorganization in the chromophore protein pocket concomitant with the formation of the intermediate T5.12. The subsequent contraction associated with a 300 ns lifetime is assigned to protein movements required to reach the entire chromoprotein free energy minimum, after the 17 ps optical decay of T5.12. The volume changes comprise the entropy of medium rearrangement during T5.12 formation and decay. The slow changes detected in previous studies by atomic force microscopy might be explained by the slowing down of movements in films containing 5,12-trans-locked bR. Photothermal beam deflection data with the 5,12-trans-locked bR suspensions indicate no further changes in microseconds to hundreds of milliseconds. Thus, all the absorbed energy is either released to the solution as heat or used for entropy changes within the first 300 ns after the pulse, supporting the paradigm that isomerization is required for signal transduction in retinal proteins. Bacterio-opsin assembled with all-trans-retinal afforded (similar to data reported with wild-type bR) an expansion of 2.6 mL/mol (assigned to the production of KE) followed by a further expansion of 0.8 mL/mol (KE-->KL; KE, KL, early and late K's) involving no heat loss. For KL decay to L, a contraction of 6 mL/mol of phototransformed reconstituted all-trans bR was determined.

A reversed-phase high-performance liquid chromatographic method to analyze retinal isomers

A high-performance liquid chromatographic (HPLC) procedure was developed to separate all- trans-, 13- cis-, 11- cis- and 9- cis-retinal isomers. Two reversed-phase Vydac C 18 columns in series were used with an isocratic solvent system of 0.1 M ammonium acetate–acetonitrile (40:60, v/v) as mobile phase and all- trans-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethyl-2,4,6,8-nonatetraene-1-ol (TMMP) as internal standard. Prior to HPLC, the retinal isomers were efficiently extracted in their original isomeric conformation using dichloromethane– n-hexane in the presence of formaldehyde. This technique is suitable for the assay of 11- cis- and all- trans-retinal isomers in retina.

Protein-Assisted Pericyclic Reactions: An Alternate Hypothesis for the Action of Quantal Receptors

The rules for allowable pericyclic reactions indicate that the photoisomerizations of retinals in rhodopsins can be formally analogous to thermally promoted Diels-Alder condensations of monoenes with retinols. With little change in the seven-transmembrane helical environment these latter reactions could mimic the retinal isomerization while providing highly sensitive chemical reception. In this way archaic progenitors of G-protein-coupled chemical quantal receptors such as those for pheromones might have been evolutionarily plagiarized from the photon quantal receptor, rhodopsin, or vice versa. We investigated whether the known structure of bacteriorhodopsin exhibited any similarity in its active site with those of the two known antibody catalysts of Diels-Alder reactions and that of the photoactive yellow protein. A remarkable three-dimensional motif of aromatic side chains emerged in all four proteins despite the drastic differences in backbone structure. Molecular orbital calculations supported the possibility of transient pericyclic reactions as part of the isomerization-signal transduction mechanisms in both bacteriorhodopsin and the photoactive yellow protein. It appears that reactions in all four of the proteins investigated may be biological analogs of the organic chemists’ chiral auxiliary-aided Diels-Alder reactions. Thus the light receptor and the chemical receptor subfamilies of the heptahelical receptor family may have been unified at one time by underlying pericyclic chemistry.

A density functional derived vibrational force field for β-ionone : Use of the ultraviolet resonance Raman intensities to check the vibrational analysis accuracy

In the present article we aim to determine an accurate and transferable molecular force field for β-ionone in order to perform molecular dynamics calculations on the retinal isomers. For β-ionone, a force field is derived from calculations using the Density Functional Theory (DFT). The force constants expressed in the internal coordinate space were scaled fitting theoretical to experimental vibrational wavenumbers. The validity of the force field was checked using a comparison between calculated and observed resonance Raman intensities obtained from the A-term part (Franck–Condon) of the scattering tensor.

Maintenance of Retinoid Metabolism in Human Retinal Pigment Epithelium Cell Culture

If transplantation of cultured retinal pigment epithelium (RPE) or iris pigment epithelium (IPE) is to be successful in the treatment of ocular disease, it is imperative to demonstrate that these cells can perform all of their necessary metabolic functions. Unfortunately, a critical function of the RPE, retinoid metabolism, is often lost rapidly in culture. We have examined whether or not nonspecific proteolytic enzymes commonly used in cell isolation and serial passaging may be responsible for this loss of function, and we have investigated novel isolation and passaging techniques which can alleviate this loss of retinoid metabolism.RPE cells were obtained from human donor eyes by enzymatic and nonenzymatic methods. Cells were cultured either on control tissue culture inserts or on inserts coated with a layer of thermally responsive poly(N -isopropylacrylamide-co-cinnamoylcarbamidemethylstyrene). Upon confluence, cells were detached either by trypsinization or by lowering dish temperature. Retinoid metabolism of cells was assessed after isolation and culture by incubating membrane fractions with3H-all- trans -retinol. Retinoid metabolism was also measured in freshly isolated IPE, corneal endothelium (CE), an RPE cell line (D407), and two hepatocyte cell lines (Hepa 6 and HepG2).Membrane fractions from cells isolated nonenzymatically or using collagenase/hyaluronidase formed 11- cis -retinol, retinal isomers and retinyl esters. Retinoid metabolism of RPE cells freshly isolated by trypsinization showed no 11- cis -retinal and little 11- cis -retinol formation. Nondamaged cells cultured on thermally responsive surfaces detached in sheets upon temperature change. They showed metabolism similar to that of cells freshly isolated by nonenzymatic means. After trypsinization, confluent cultures dissociated into individual cells, but these cells showed poor retinoid metabolism, including no detectable retinyl esters or 11-cis -retinoid isomers. IPE, CE and Hepa 6 did not show any retinoid metabolism. D407 and HepG2 produced retinals, but not the 11- cis isomer.RPE cells isolated using trypsin lose the ability to form critical intermediates in the visual cycle. Collagenase/hyaluronidase or nonenzymatic cell isolation techniques enable these functions to be maintained. After cell culture, thermally responsive surfaces allow nonenzymatic cell detachment and excellent maintenance of retinoid metabolism.

Kinetic properties of the human liver cytosolic aldehyde dehydrogenase for retinal isomers

Retinoic acid exerts pleiotropic effects by acting through two families of nuclear receptors, RAR and RXR. All- trans and 9- cis retinoic acid bind RARs, whereas 9- cis retinoic acid binds and activates only the RXRs. To understand the role of human liver cytosolic aldehyde dehydrogenase (ALDH1) in retinoic acid synthesis, we examined the ability of ALDH1 to catalyze the oxidation of the naturally occurring retinal isomers. ALDH1 catalyzed the oxidation of all- trans, 9- cis, and 13- cis retinal with equal efficiency. However, the affinity to all- trans retinal (K m = 2.2 μM) was twofold higher than to 9- cis (K m = 5.5 μM) and 13- cis (K m = 4.6μM) retinal. All- trans retinol was a potent inhibitor of ALDH1 activity, and inhibited all- trans retinal oxidation uncompetitively. Comparison of the kinetic properties of ALDH1 for retinal isomers with those of previously reported rat kidney retinal dehydrogenase showed distinct differences, suggesting that ALDH1 may play a different role in retinal metabolism in liver.

Vibrational relaxation during the retinal isomerization in Bacteriorhodopsin

Femtosecond time-resolved optical pump–infrared probe spectroscopy was employed to characterize the ethylenic (CC)-stretch vibrational dynamics (1498–1542 cm −1) of the retinal chromophore in Bacteriorhodopsin (BR) during the initial, all-trans to 13-cis isomerization. The early branching reaction was observed, i.e. formation of the ground-state 13-cis product, K, and partial recovery of the all-trans educt state BR 570. The BR 570 recovery occurs within 2 ps and thus is faster than K-formation (3–4 ps). The IR transients are described in terms of a kinetic model involving vibrational precursors for K and for the recovering BR 570, respectively.

Quantitative Determination of the Protein Catalytic Efficiency for the Retinal Excited-State Decay in Bacteriorhodopsin

It was previously found that by removing the negative charge of Asp85 in bacteriorhodopsin (bR), either by protonating it (as in deionized bR) or by mutation to Asn, the decay time of the retinal excited state increases from 0.5 ps to either 1.5 or 10 ps. The two decay components result from the presence of all-trans and 13-cis,15-syn (13-cis) retinal isomers in the modified retinal protein. To quantitatively determine the protein catalysis for the primary process in native bR, we need to determine which decay component results from the excited state of the all-trans isomer (present in the native bR). It is known that the all-trans isomer absorbs at longer wavelength than the 13-cis isomer in blue bR. In this communication, we report the results of pump−probe experiments using 100 fs laser pulses. Probing is carried out at 490 nm, where the excited state in both isomers absorbs. It is found that the ratio of the amplitudes of the two decay components in blue bR changes with variation of the excitation wav...

Assessing the functionality of a membrane protein in a three-dimensional crystal

Hexagonal microcrystals of bacteriorhodopsin embedded in a lipidic cubic phase have been investigated by time-resolved FT-IR and resonance Raman spectroscopy. Retinal isomerization, conformational changes in the protein backbone and proton translocation are virtually indistinguishable from those in the native membrane. The protein is thus fully active in three-dimensional crystals.

Conformational changes in the core structure of bacteriorhodopsin.

Bacteriorhodopsin (bR) is the light-driven proton pump found in the purple membrane of Halobacterium salinarium. In this work, structural changes occurring during the bR photocycle in the core structure of bR, which is normally inaccessible to hydrogen/deuterium (H/D) exchange, have been probed. FTIR difference bands due to vibrations of peptide groups in the core region of bR have been assigned by reconstituting and regenerating delipidated bR in the presence of D2O. Exposure of bR to D2O even after long periods causes only a partial shift of the amide II band due to peptide NH --> ND exchange only of peripheral peptide structure. However, the amide II band completely downshifts when reconstitution/regeneration of bR is performed in the presence of D2O, indicating that almost the entire core backbone structure of bR undergoes H/D exchange. Peripheral regions can then be reexchanged in H2O, leaving the core backbone region deuterated. Low-temperature FTIR difference spectra on these core-deuterated samples reveal that peptide groups in the core region respond to retinal isomerization as early as the K intermediate. By formation of the M intermediate, infrared differences in the amide I region are dominated by much larger structural changes occurring in the core structure. In the amide II region, difference bands appear upon K formation and increase upon M formation which are similar to those observed upon the cooling of bacteriorhodopsin. This work shows that retinal isomerization induces conformational changes in the bacteriorhodopsin core structure during the early photocycle which may involve an increase in the strength of intramolecular alpha-helical hydrogen bonds.

Single-run analysis of retinal isomers, retinol and photooxidation products by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) procedure was developed to allow the rapid separation, in a single run, of a mixture of the main retinal isomers (all- trans, 13- cis, 9- cis), all- trans-retinol, and of the two major photooxygenated photoproducts (5,8-peroxyretinal and 5,6-epoxyretinal). The mixture was separated by HPLC on an octadecyl (ODS) column with 16% (v/v) diethyl ether in hexane as mobile phase and anthracene as the internal standard. A commercial type cosmetic formulation containing 0.05% all- trans-retinal was analyzed successfully for this analyte.

Free-energy simulations of the retinal cis --> trans isomerization in bacteriorhodopsin.

Free-energy profiles for ground-state cis --> trans isomerization of retinal in vacuum, in solution, and in the protein bacteriorhodopsin are calculated using free-energy simulations. The free-energy barriers in the protein were 9 kcal/mol for ionized Asp85 and 14 kcal/mol for neutral Asp85, significantly lower than those found in solution (18 kcal/mol) or vacuum (19 kcal/mol). Therefore, bacteriorhodopsin can be said to act as a catalyst in the isomerization. The barrier in the protein is due mainly to stabilization of the transition state through favorable nonbonded interactions with the protein part of the system, with internal strain and interactions with solvent playing minor roles. The protonated Asp85 simulation models the behavior of the system in the N --> O transition. Our calculated 14 kcal/mol barrier and 4-ms relaxation time for this process are in excellent agreement with experimentally measured values of 12 kcal/mol and 5 ms, respectively. The ionized Asp85 simulation models two hypothetical processes: the N --> O transition with a proton removed from Asp85 and the initial BR568 --> L transition on the ground-state energy surface. The cis-trans isomerization barrier in this system is 9 kcal/mol, the lowest of all the studied cases. The presence of the charged carboxylate group in the ionized Asp85 system leads to strong stabilization of the transition state by interactions with the surroundings and changes the distance between Asp85 and the Schiff base proton compared to the corresponding distance in the neutral Asp85 system. This suggests that the protonation of Asp85 plays an important role in regulating access to the Schiff base proton. For both Asp85 ionization states the calculated cis-trans free-energy difference was close to 0, indicating that the protein can accommodate both retinal isomers equally well. The computed negligible difference between the N and O free-energy levels is in accord with experimental data.

Cl −-dependent photovoltage responses of bacteriorhodopsin: comparison of the D85T and D85S mutants and wild-type acid purple form

Laser flash-induced photovoltage responses of the D85S and D85T mutants as well as of the wild-type acid blue form are similar and reflect intraprotein charge redistribution caused by retinal isomerization. The Cl −-induced transition of all of these blue forms into purple ones is accompanied by the appearance of electrogenic stages, which is probably associated with Cl − translocation in the cytoplasmic direction. Cl − translocation efficiency of these purple forms is much lower than that of the proton transport by the wild-type bacteriorhodopsin. The values of the efficiency do not exceed 15, 8 and 3% for the D85T, D85S and wild-type acid purple form, respectively. Cl − induces an additional electrogenic phase in the photovoltage responses of the D85S mutant and the wild-type acid purple form. This phase is supposed to be associated with the reversible Cl − movement in the extracellular direction. It is interesting that this component is absent in the photovoltage response of the D85T mutant which has, like halorhodopsin, a threonine residue at position 85.

Direct Measurements for Isomer Ratio of Retinal in Bacteriorhodopsin by an Electrochemical Method

Abstract— The composition of retinal isomers in bacteriorhodopsin (bR) in purple membrane (PM) was determined by photoelectric response measurements using a sandwich‐type electrochemical cell. The measured amplitude of the photocurrent obtained from a dark‐adapted sample was 55% lower than that from a light‐adapted sample. This ratio, 55:45, would correspond to the 13‐cis/aU‐trans isomer ratio of retinal in the dark if the 13‐cis form of the pigment did not give a response. This amplitude change correlated with the visible spectral shift of bR. The isomer ratio in the dark depended only weakly on the temperature of the electrolyte, whereas the retinal isomerization rate strongly depended on the temperature and the pH of the electrolyte in the cell. Our results indicate that photoelectric response is elicited only by a species originating from bR containing all‐trans retinal and that the behavior of the response in the dark is associated with the pKa of the proton release kinetics of Asp‐85.

Ultraviolet resonance Raman examination of the light-induced protein structural changes in rhodopsin activation.

Ultraviolet resonance Raman (UVRR) spectra of rhodopsin and its metarhodopsin I and metarhodopsin II photointermediates have been obtained to examine the molecular mechanism of G-protein-coupled receptor activation. Spectra were acquired using a single-pass capillary flow technique in combination with a Littrow prism UV prefilter detection system. The UVRR difference spectra between rhodopsin and mearhodopsin I exhibit small differences assignalbe to tyrosine residues and no differences due to tryptophan. The UVRR difference spectra between rhodopsin and metarhodopsin II exhibit significant differences for vibrations of both tryptophan and tyrosine residues. Most importantly, there is and intensity decrease of the totally symmetric tryptophan modes at 759, 1008, and 1545 cm-1, an intensity decrease of the tryptophan W7 band at 1357 cm-1, and a frequency shift of the tryptophan W17 ban from 885 to 892 cm-1. These difference features are assigned to one or more tryptophan residues that reside in a hydrophobic, weakly hydrogen-bonding environment in rhodopsin and that are transferred to a less hydrophobic, non-hydrogen-bonding environment during rhodopsin activation. The available evidence suggests that Trp265 makes a dominant contribution to the tryptophan features in this difference spectrum. These results are interpreted with a model for rhodopsin activation in which retinal isomerization alters the interaction of Trp265 with the ionone ring of the retinal chromophore.

Photoinitiated electron transfer interaction of all-trans retinal with electron donors and acceptors

Formation of radical ions of all-trans retinal (ATR) has been demonstrated using 1H CIDNP on its photoinitiated interaction with electron donor (stilbene) and acceptors (2,5-dichlorobenzoquinone, chloranil). CIDNP data show qualitative agreement with calculations of spin density distribution in ATR radical ions conducted by means of the INDO/AM1 method. It has been found that CIDNP effects are formed in the reaction of reversible electron transfer between ATR and either electron donors or acceptors. Retinal isomers arising under irradiation also give rise to nonequilibrium population of spin states in NMR spectra. The correlation between CIDNP effects and the processes of cis-trans isomerization has been discussed.

The steric trigger in rhodopsin activation

Rhodopsin is the seven transmembrane helix receptor responsible for dim light vision in vertebrate rod cells. The protein has structural homology with the other G protein-coupled receptors, which suggests that the tertiary structures and activation mechanisms are likely to be similar. However, rhodopsin is unique in several respects. The most striking is the fact that the receptor “ligand”, 11-cis retinal, is covalently bound to the protein and is converted from an “antagonist” to an “agonist” upon absorption of light. NMR studies of rhodopsin and its primary photoproduct, bathorhodopsin, have generated structural constraints that enabled docking of the 11-cis and all-trans retinal chromophores into a low-resolution model of the protein proposed by Baldwin. These studies also suggest a mechanism for how retinal isomerization leads to rhodopsin activation. More recently, mutagenesis studies have extended these results by showing how the selectivity of the retinal-binding site can be modified to favor the all-trans over the 11-cis isomer. The structural constraints produced from these studies, when placed in the context of a high-resolution model of the protein, provide a coherent picture of the activation mechanism, which we show involves a direct steric interaction between the retinal chromophore and transmembrane helix 3 in the region of Gly121.