Chromophore In Bacteriorhodopsin Research Articles

Protein−β-Ionone Ring Interactions Enhance the Light-Induced Dipole of the Chromophore in Bacteriorhodopsin

Following light absorption, the retinal chromophore of bacteriorhodospin experiences very large dipolar changes in the vertically excited state. This light-induced dipole is at least 50% larger than that of the retinal chromophore in films or in solution. We have studied the origin of the protein effect by applying second harmonic generation measurements of artificial bacteriorhodopsin pigments derived from synthetic retinal analogues, characterized by a modified polyene chain length and ring−chain conformation. The studies demonstrated a significant influence of a protein domain in the vicinity of the retinal β-ionone. We suggest that tryptophane residues (specifically Trp 138 and 189) enhance the light-induced dipole in bacteriorhodopsin. The data also point to another protein domain (Trp 182) influencing the retinal chromophore. The effect of these tryptophane residues appears not only to stabilize the light-induced charge redistribution but also to enable the migration in the excited state of the posi...

The interaction between purple membrane and membrane lipid

Bacteriorhodopsin in purple membrane was reconstituted into different lipid vesicles. The effect of three different lipids on the structure and function of bacteriorhodopsin in lipid vesicles was studied by the observation on freeze-fracture eletron microscopy, the rotational diffusion of bacteriorhodopsin in lipid vesicles, the measurement of absorption spectrum, and the absorbance change with time. For these prepared samples, the results showed that DMPC was the stable lipid environment of bacteriorhodopsin; egg-pc causeed the loss of retinal chromophore of bacteriorhodopsin and it was not reversible change, cholesterol could stabilize the bacteriorhodopsin in lipid environment, but it caused the aggregation of bacteriorhodopsin.

Real-time spectroscopy of transition states in bacteriorhodopsin during retinal isomerization.

Real-time investigations of the rearrangement of bonds during chemical transformations require femtosecond temporal resolution, so that the atomic vibrations within the reacting molecules can be observed. Following the development of lasers capable of emitting ultrashort laser flashes on this timescale, chemical reactions involving relatively simple molecules have been monitored in detail, revealing the transient existence of intermediate species as reactants are transformed into products. Here we report the direct observation of nuclear motion in a complex biological system, the retinal chromophore of bacteriorhodopsin (bR568), as it undergoes the trans-cis photoisomerization that is fundamental to the vision process. By using visible-light pulses of less than 5 femtosecond in duration, we are able to monitor changes in the vibrational spectra of the transition state and thus show that despite photoexcitation of the anti-bonding molecular orbital involved, isomerization does not occur instantly, but involves transient formation of a so-called 'tumbling state'. Our observations thus agree with growing experimental and ab initio evidence for a three-state photoisomerization model and firmly discount the initially suggested two-state model for this process.

Computational evidence in favor of a two-state, two-mode model of the retinal chromophore photoisomerization.

In this paper we use ab initio multiconfigurational second-order perturbation theory to establish the intrinsic photoisomerization path model of retinal chromophores. This is accomplished by computing the ground state (S(0)) and the first two singlet excited-state (S(1), S(2)) energies along the rigorously determined photoisomerization coordinate of the rhodopsin chromophore model 4-cis-gamma-methylnona-2,4,6,8-tetraeniminium cation and the bacteriorhodopsin chromophore model all-trans-hepta-2,4, 6-trieniminium cation in isolated conditions. The computed S(2) and S(1) energy profiles do not show any avoided crossing feature along the S(1) reaction path and maintain an energy gap >20 kcal small middle dotmol(-1). In addition, the analysis of the charge distribution shows that there is no qualitative change in the S(2) and S(1) electronic structure along the path. Thus, the S(1) state maintains a prevalent ionic (hole-pair) character whereas the S(2) state maintains a covalent (dot-dot) character. These results, together with the analysis of the S(1) reaction coordinate, support a two-state, two-mode model of the photoisomerization that constitutes a substantial revision of the previously proposed models.

Characterization of a conical intersection between the ground and first excited state for a retinal analog

Ab initio complete active space SCF calculations have been carried out to investigate the first excited electronic state of a retinal protonated Schiff base analog: all-trans-3,7-dimethylnona-2,4,6,8-tetraenmethylimminium cation. This model of the retinal chromophore in bacteriorhodopsin includes five conjugated double bonds as well as both pertinent backbone methyl groups. The excited state minimum that is relevant for isomerization in bacteriorhodopsin is investigated and is found to be in very close proximity to a Jahn–Teller conical intersection. The two (global) coordinates that are most effective in promoting efficient internal conversion back to the ground electronic state (by lifting the degeneracy between the ground and first excited state) are identified and discussed, and the distribution of the positive charge in the retinal analog as a function of these two coordinates is investigated.

Molecular Dynamics Study of the Nature and Origin of Retinal's Twisted Structure in Bacteriorhodopsin

The planarity of the polyene chain of the retinal chromophore in bacteriorhodopsin is studied using molecular dynamics simulation techniques and applying different force-field parameters and starting crystal structures. The largest deviations from a planar structure are observed for the C 13 C 14 and C 15 N 16 double bonds in the retinal Schiff base structure. The other dihedral angles along the polyene chain of the chromophore, although having lower torsional barriers in some cases, do not significantly deviate from the planar structure. The results of the simulations of different mutants of the pigment show that, among the studied amino acids of the binding pocket, the side chain of Trp-86 has the largest impact on the planarity of retinal, and the mutation of this amino acid to alanine leads to chromophore planarity. Deletion of the methyl C 20, removal of a water molecule hydrogen-bonded to H 15, or mutation of other amino acids to alanine did not show any significant influence on the distortion of the chromophore. The results from the present study suggest the importance of the bulky residue of Trp-86 in the isomerization process, in both ground and excited states of the chromophore, and in fine-tuning of the p K a of the retinal protonated Schiff base in bacteriorhodopsin. The dark adaptation of the pigment and the last step of the bacteriorhodopsin photocycle imply low barriers against the rotation of the double bonds in the Schiff base region. The twisted double bonds found in the present study are consistent with the proposed mechanism of these ground state isomerization events.

The angles between the C(1)-, C(5)-, and C(9)-methyl bonds of the retinylidene chromophore and the membrane normal increase in the M intermediate of bacteriorhodopsin: direct determination with solid-state (2)H NMR.

The orientations of three methyl bonds of the retinylidene chromophore of bacteriorhodopsin were investigated in the M photointermediate using deuterium solid-state NMR ((2)H NMR). In this key intermediate, the chromophore has a 13-cis, 15-anti conformation and a deprotonated Schiff base. Purple membranes containing wild-type or mutant D96A bacteriorhodopsin were regenerated with retinals specifically deuterated in the methyl groups of either carbon C(1) or C(5) of the beta-ionone ring or carbon C(9) of the polyene chain. Oriented hydrated films were formed by drying concentrated suspensions on glass plates at 86% relative humidity. The lifetime of the M state was increased in the wild-type samples by applying a guanidine hydrochloride solution at pH 9.5 and in the D96A sample by raising the pH. (2)H NMR experiments were performed on the dark-adapted ground state (a 2:1 mixture of 13-cis, 15-syn and all-trans, 15-anti chromophores), the cryotrapped light-adapted state (all-trans, 15-anti), and the cryotrapped M intermediate (13-cis, 15-anti) at -50 degrees C. Bacteriorhodopsin was first completely converted to M under steady illumination of the hydrated films at +5 degrees C and then rapidly cooled to -50 degrees C in the dark. From a tilt series of the oriented sample in the magnetic field and an analysis of the (2)H NMR line shapes, the angles between the individual C-CD(3) bonds and the membrane normal could be determined even in the presence of a substantial degree of orientational disorder. While only minor differences were detected between dark- and light-adapted states, all three angles increase in the M state. This is consistent with an upward movement of the C(5)-C(13) part of the polyene chain toward the cytoplasmic surface or with increased torsional strain. The C(9)-CD(3) bond shows the largest orientational change of 7 degrees in M. This reorientation of the chromophore in the binding pocket provides direct structural support for previous suggestions (based on spectroscopic evidence) for a steric interaction in M between the C(9)-methyl group and Trp 182 in helix F.

Structural change of threonine 89 upon photoisomerization in bacteriorhodopsin as revealed by polarized FTIR spectroscopy.

The all-trans to 13-cis photoisomerization of the retinal chromophore of bacteriorhodopsin occurs selectively, efficiently, and on an ultrafast time scale. The reaction is facilitated by the surrounding protein matrix which undergoes further structural changes during the proton-transporting reaction cycle. Low-temperature polarized Fourier transform infrared difference spectra between bacteriorhodopsin and the K intermediate provide the possibility to investigate such structural changes, by probing O-H and N-H stretching vibrations [Kandori, Kinoshita, Shichida, and Maeda (1998) J. Phys. Chem. B 102, 7899-7905]. The measurements of [3-18O]threonine-labeled bacteriorhodopsin revealed that one of the D2O-sensitive bands (2506 cm(-1) in bacteriorhodopsin and 2466 cm(-1) in the K intermediate, in D2O exhibited 18(O)-induced isotope shift. The O-H stretching vibrations of the threonine side chain correspond to 3378 cm(-1) in bacteriorhodopsin and to 3317 cm(-1) in the K intermediate, indicating that hydrogen bonding becomes stronger after the photoisomerization. The O-H stretch frequency of neat secondary alcohol is 3340-3355 cm(-1). The O-H stretch bands are preserved in the T46V, T90V, T142N, T178N, and T205V mutant proteins, but diminished in T89A and T89C, and slightly shifted in T89S. Thus, the observed O-H stretching vibration originates from Thr89. This is consistent with the atomic structure of this region, and the change of the S-H stretching vibration of the T89C mutant in the K intermediate [Kandori, Kinoshita, Shichida, Maeda, Needleman, and Lanyi (1998) J. Am. Chem. Soc. 120, 5828-5829]. We conclude that all-trans to 13-cis isomerization causes shortening of the hydrogen bond between the OH group of Thr89 and a carboxyl oxygen atom of Asp85.

Role of Isomerization Barriers in the pKa Control of the Retinal Schiff Base: A Density Functional Study

The barriers to the rotation of all conventional single and double bonds have been calculated in a model Schiff base which is structurally very close to the retinal Schiff base chromophore in bacteriorhodopsin (bR). The calculated values for the torsional barriers can be used, therefore, to estimate the corresponding values in the retinal Schiff base structure. The torsional barriers are calculated as the energy differences between the all-trans and 90°-rotated conformers, respectively, for optimized structures in both neutral and protonated species. To study the effect of the isomerization on the pKa of the Schiff base, for each 90°-rotated conformer the gas-phase proton affinity of the system has also been calculated and compared with the proton affinity of the all-trans conformer. The results clearly show that protonation of the nitrogen in the Schiff base group has a profound effect on the barriers to the rotation around different bonds in the system. For 90°-twisted species, completely different mesomeric structures are predicted for single or double bond rotations as well as for the protonated and the neutral species. The CN double bond and the double bond next to the Schiff base group in the protonated Schiff base (corresponding to the C13C14 bond of the retinal Schiff base in the bR which is assumed to rotate during the photoisomerization) have the lowest isomerization barriers among the double bonds. In the neutral species, however, high barriers to these rotations are predicted. The proton affinity of the system decreases upon the isomerization around the single bonds, while the rotation around the double bonds significantly increases the calculated values for the proton affinities. This may be considered as one of the possible mechanisms by which the protein environment controls the pKa of the retinal Schiff base chromophore. The predicted low barrier to the C13C14 double bond rotation is needed for the ground-state isomerization of the chromophore in the last step of the bR photocycle and also in the dark adaptation of the pigment. Protonation of the retinal Schiff base chromophore, therefore, seems to be a prerequisite for these ground-state isomerization events in bR.

Angular Orientation of the Retinyl Chromophore of Bacteriorhodopsin: Reconciliation of 2H NMR and Optical Measurements

It is argued on the basis of MNDO−PSDCI calculations that, at the wavelength of electronic dichroism studies used to infer structure, the transition dipole moment of the retinyl Schiff's base chromophore of bacteriorhodopsin makes an angle of 10.5 ± 3.5° with respect to the long axis of the chromophore. The magnitude and direction of this off-axis angle helps to reconcile the differences between the deuterium NMR and optical determinations of the angular orientation of the chromophore in bacteriorhodopsin. Reconciliation of the NMR and optical dichroism structures is only possible, however, when the chromophore is oriented so that the imine proton points toward the extracellular surface. After correction, the published electronic dichroism data predict a chromophore angle of Ω = 61 ± 7°, where Ω is defined as the angle of a line connecting chromophore atoms C5 and C15 relative to the membrane normal. The corresponding NMR results are Ω = 53.7 ± 4.1°. The combined data with equal weighting yield Ω = 57 ± 8...

Temperature Effects Of Excitation Laser Pulses During Step-Scan FT-IR Measurements

Replacing the chromophore of bacteriorhodopsin with chemically modified retinal analogs which cannot isomerize, allows to measure directly side effects from the laser pulse used for sample excitation in step-scan FT-IR measurements. Comparison with static temperature difference spectra and temperature-jump experiments shows that the observed effects can mainly be attributed to heating of the sample by the laser pulse. The size of resulting spectral changes is compared to difference bands of the photoreaction.

1PB018 バクテリオロドプシンの光異性化反応におけるThr89の構造変化

The all-trans to 13-cis photoisomerization of the retinal chromophore of bacteriorhodopsin occurs selectively, efficiently, and on an ultrafast time scale. The reaction is facilitated by the surrounding protein matrix which undergoes further structural changes during the proton-transporting reaction cycle. Low-temperature polarized Fourier transform infrared difference spectra between bacteriorhodopsin and the K intermediate provide the possibility to investigate such structural changes, by probing O−H and N−H stretching vibrations [Kandori, Kinoshita, Shichida, and Maeda (1998) J. Phys. Chem. B 102, 7899−7905]. The measurements of [3-18O]threonine-labeled bacteriorhodopsin revealed that one of the D2O-sensitive bands (2506 cm-1 in bacteriorhodopsin and 2466 cm-1 in the K intermediate, in D2O) exhibited 18O-induced isotope shift. The O−H stretching vibrations of the threonine side chain correspond to 3378 cm-1 in bacteriorhodopsin and to 3317 cm-1 in the K intermediate, indicating that hydrogen bonding be...

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.

Ab initio MO study on the potential energy surfaces of low-lying excited states of protonated Schiff base retinal

The potential energy surface of the protonated Schiff base of retinal has been calculated at the state-average complete active space self-consistent field (sa-CASSCF) level of theory to elucidate the mechanism of the trans– cis photoisomerization of the chromophore in bacteriorhodopsin. We treated two molecular systems, i.e., protonated N-methyl Schiff base retinal (C 21H 32N +) and its simplified model compound, C 12H 16N +. We used the 6-31g split-valence basis set. To clarify the effect of the protein and hydrogen bonding with water, the same molecular system with a negative point charge or with a water molecule in the vicinity of the Schiff base was also calculated. The results show that the electrostatic environment of the protein works to produce a favorable potential energy surface for the lowest excited state of the chromophore, leading to efficient trans– cis photoisomerization.

Chromophore orientation in bacteriorhodopsin determined from the angular dependence of deuterium nuclear magnetic resonance spectra of oriented purple membranes.

The orientation of prosthetic groups in membrane proteins is of considerable importance in understanding their functional role in energy conversion, signal transduction, and ion transport. In this work, the orientation of the retinylidene chromophore of bacteriorhodopsin (bR) was investigated using 2H NMR spectroscopy. Bacteriorhodopsin was regenerated with all-trans-retinal stereospecifically deuterated in one of the geminal methyl groups on C1 of the cyclohexene ring. A highly oriented sample, which is needed to obtain individual bond orientations from 2H NMR, was prepared by forming hydrated lamellar films of purple membranes on glass slides. A Monte Carlo method was developed to accurately simulate the 2H NMR line shape due to the distribution of bond angles and the orientational disorder of the membranes. The number of free parameters in the line shape simulation was reduced by independent measurements of the intrinsic line width (1.6 kHz from T2e experiments) and the effective quadrupolar coupling constant (38. 8-39.8 kHz from analysis of the line shape of a powder-type sample). The angle between the C1-(1R)-1-CD3 bond and the purple membrane normal was determined with high accuracy from the simultaneous analysis of a series of 2H NMR spectra recorded at different inclinations of the uniaxially oriented sample in the magnetic field at 20 and -50 degrees C. The value of 68.7 +/- 2.0 degrees in dark-adapted bR was used, together with the previously determined angle of the C5-CD3 bond, to calculate the possible orientations of the cyclohexene ring in the membrane. The solutions obtained from 2H NMR were then combined with additional constraints from linear dichroism and electron cryomicroscopy to obtain the allowed orientations of retinal in the noncentrosymmetric membrane structure. The combined data indicate that the methyl groups on the polyene chain point toward the cytoplasmic side of the membrane and the N-H bond of the Schiff base to the extracellular side, i.e., toward the side of proton release in the pump pathway.

Refolding of Bacteriorhodopsin from Expressed Polypeptide Fragments

Bacteriorhodopsin is a heptahelical membrane protein that can be refolded to the native state following denaturation. To analyze the in vitro folding process with independent structural domains, eight fragments comprising two (AB, FG), three (AC, EG), four (AD, DG) or five (AE, CG) of the transmembrane segments were produced by expression in Escherichia coli. The polypeptides were purified to homogeneity by solvent extraction of E. coli membranes, repeated phase separation, and anion-exchange chromatography employing the C-terminal tail of bacteriorhodopsin for adsorption. Upon reconstitution into phospholipid/detergent micelles pairs of complementary fragments (AB.CG, AC.DG, AD.EG, and AE.FG) assembled in the presence of retinal to regenerate the characteristic bacteriorhodopsin chromophore with high efficiency. Together with previous studies, these results demonstrate that the covalent connections in each of the six interhelical loops are dispensable for a correct association of the helices. The different loops, however, contribute to the stability of the folded structure, as shown by increased susceptibilities toward denaturation in SDS and at acidic pH, and decreased Schiff base pKa values for the AB.CG, AC. DG, AD.EG, and AE.FG complexes, compared with the intact protein. Notably, the heptahelical bundle structure was also generated by all possible combinations of pairs of overlapping fragments, containing one (AC.CG, AD.DG, AE.EG), two (AD.CG, AE.DG), or three (AE.CG) redundant helices. The spectral properties of the chromophores indicate that the retinal-binding pocket of the AC.CG, AD.CG, and AE. CG complexes is formed by helices A and B of the respective N-terminal fragment and the C-terminal CG fragment, whereas the AD. DG, AE.DG, and AE.EG complexes are likely to adopt a heptahelical bundle structure analogous to AD.EG. The combined data show that the specificity of the helix assembly of bacteriorhodopsin is influenced by connectivities provided by the C-D and E-F surface loops.

Similarity of bacteriorhodopsin structural changes triggered by chromophore removal and light-driven proton transport

Bacteriorhodopsin (bR) is the light-driven proton pump found in the purple membrane of Halobacterium salinarium. A series of conformational changes occur during the bR photocycle which involve alterations in buried-helical structure as well as in the protonation state of Asp residues which are part of the proton transport pathway. Here we report evidence that similar conformational changes occur upon removal of the retinylidene chromophore of bacteriorhodopsin to form the apoprotein bacterioopsin (bO). This suggests a simple ligand-binding model of proton transport in bacteriorhodopsin which may have relevance to other transport and signal transducing membrane proteins including the visual photoreceptor rhodopsin.

Expression of bacteriorhodopsin in Sf9 and COS-1 cells.

We report studies on the expression of the archaebacterial membrane protein bacteriorhodopsin in Sf9 insect cells and in COS-1 mammalian cells. In both cell systems, the apoprotein bacterio-opsin was expressed at levels of approximately 1 microgram/10(6) cells. Immunofluorescence studies showed that the expressed protein was accumulated in the endoplasmic reticulum. However, upon addition of all-trans retinal to membranes isolated from either Sf9 or COS-1 cells expressing bacterio-opsin, the characteristic bacteriorhodopsin chromophore (lambda max at approximately 560 nm) was rapidly generated. This is in contrast to bacterio-opsin expressed in E. coli, which cannot be functionally reconstituted with retinal unless it is first denatured, and then renatured in vitro. These studies demonstrate that the bacterio-opsin expressed is correctly folded and show that localization of a heterologously expressed membrane protein in the endoplasmic reticulum does not necessarily imply that it is misfolded.

Miscellanea. Femtosecond infrared spectroscopy on bacteriorhodopsin using a broad band carbon monoxide laser

AbstractA pump‐probe mid infrared transient absorption apparatus was built, employing a broad band continuous wave carbon monoxide laser and an optical gating technique resulting in femtosecond time resolution.It was used to monitor the all‐trans to 13‐cis isomerization of the retinal chromophore in Bacteriorhodopsin (BR). The C=C in‐phase stretch vibration, located at 1529cm−1 in the light adapted, unphotolyzed state BR570 served as marker band. Photoexcitation leads to a bleach centered at this wavenumber and to a partial recovery corresponding to the non unity quantum yield of the BR photocycle. On the picosecond time scale the high energy shoulder of this bleach is superimposed by newly created IR absorption strength, due to the corresponding vibrational modes of the J‐ and the K‐state.

The synthesis of a coumarin analogue of retinal

A new coumarin analogue of retinal was synthesised by Wittig condensation of (2-oxo-2H-chromen-3-yl)methyltriphenylphosphonium chloride and (2E,6Z) 2,6-dimethyl-8-triphenylsilyloxyocta-2,6-dien-4-yn-l-al in high yield. Subsequent triphenylsilyl group removal, MnO 2 oxidation, followed by hydrogenation over Lindlar catalyst and isomerization in the presence of traces of iodine furnished the desired polyenal. This all-trans coumarin analogue of retinal was used for study of the bacteriorhodopsin chromophore binding site.