Retinal Chromophore In Bacteriorhodopsin Research Articles

Born–Oppenheimer molecular dynamics study on collective protein dynamics invoked by ultrafast photoisomerization of retinal chromophore in bacteriorhodopsin

Large-scale quantum-chemistry-based Born–Oppenheimer molecular dynamics simulations of collective protein dynamics of bacteriorhodopsin initiated by photoinduced all-trans→ 13-cis isomerization of retinal Schiff base chromophore compensate for missing information from time-resolved serial femtosecond X-ray crystallography measurements in terms of electronic states, energy transfer, and details of structural dynamics.

Protein Dynamics Preceding Photoisomerization of the Retinal Chromophore in Bacteriorhodopsin Revealed by Deep-UV Femtosecond Stimulated Raman Spectroscopy.

Bacteriorhodopsin is a prototypical photoreceptor protein that functions as a light-driven proton pump. The retinal chromophore of bacteriorhodopsin undergoes C13═C14 trans-to-cis isomerization upon photoexcitation, and it has been believed to be the first event that triggers the cascaded structural changes in bacteriorhodopsin. We investigated the protein dynamics of bacteriorhodopsin using deep-ultraviolet resonance femtosecond stimulated Raman spectroscopy. It was found that the stimulated Raman signals of tryptophan and tyrosine residues exhibit significant changes within 0.2 ps after photoexcitation while they do not noticeably change during the isomerization process. This result implies that the protein environment changes first, and its change is small during isomerization. The obtained femtosecond stimulated Raman data indicate that ultrafast change is induced in the protein part by the sudden creation of the large dipole of the excited-state chromophore, providing an environment that realizes efficient and selective isomerization.

Tip-Enhanced Infrared Difference-Nanospectroscopy of the Proton Pump Activity of Bacteriorhodopsin in Single Purple Membrane Patches.

Photosensitive proteins embedded in the cell membrane (about 5 nm thickness) act as photoactivated proton pumps, ion gates, enzymes, or more generally, as initiators of stimuli for the cell activity. They are composed of a protein backbone and a covalently bound cofactor (e.g. the retinal chromophore in bacteriorhodopsin (BR), channelrhodopsin, and other opsins). The light-induced conformational changes of both the cofactor and the protein are at the basis of the physiological functions of photosensitive proteins. Despite the dramatic development of microscopy techniques, investigating conformational changes of proteins at the membrane monolayer level is still a big challenge. Techniques based on atomic force microscopy (AFM) can detect electric currents through protein monolayers and even molecular binding forces in single-protein molecules but not the conformational changes. For the latter, Fourier-transform infrared spectroscopy (FTIR) using difference-spectroscopy mode is typically employed, but it is performed on macroscopic liquid suspensions or thick films containing large amounts of purified photosensitive proteins. In this work, we develop AFM-assisted, tip-enhanced infrared difference-nanospectroscopy to investigate light-induced conformational changes of the bacteriorhodopsin mutant D96N in single submicrometric native purple membrane patches. We obtain a significant improvement compared with the signal-to-noise ratio of standard IR nanospectroscopy techniques by exploiting the field enhancement in the plasmonic nanogap that forms between a gold-coated AFM probe tip and an ultraflat gold surface, as further supported by electromagnetic and thermal simulations. IR difference-spectra in the 1450–1800 cm–1 range are recorded from individual patches as thin as 10 nm, with a diameter of less than 500 nm, well beyond the diffraction limit for FTIR microspectroscopy. We find clear spectroscopic evidence of a branching of the photocycle for BR molecules in direct contact with the gold surfaces, with equal amounts of proteins either following the standard proton-pump photocycle or being trapped in an intermediate state not directly contributing to light-induced proton transport. Our results are particularly relevant for BR-based optoelectronic and energy-harvesting devices, where BR molecular monolayers are put in contact with metal surfaces, and, more generally, for AFM-based IR spectroscopy studies of conformational changes of proteins embedded in intrinsically heterogeneous native cell membranes.

Fragment-based configuration interaction wave function to calculate environmental effect on excited states in proteins and solutions

• Coarse-grained analysis to figure out excited-state w.f. including environments. • A fragment-based truncated CIS for the excited-state w.f. including environments. • CTs between environmental fragments give negligible contributions. Solvatochromic effect in proteins and solutions was described by a configuration interaction singles (CIS) wave function with fragment-localized molecular orbitals. Coarse-grained analysis indicated that the CI wave function can be described by local excitations and charge-transfer (CT) excitations between the chromophore and the environment. We developed an atomic-orbital direct runcated CIS code and applied the excited states of retinal chromophore in bacteriorhodopsin and MeOH environments, and those of s -trans-acrolein in water. Number of excitation operators was significantly reduced by eliminating the CT excitations between the environmental fragments. The truncated CIS wave functions reproduced the original excitation energies very well.

Ground‐ and Excited‐State Vibrational Coherence Dynamics in Bacteriorhodopsin Probed With Degenerate Four‐Wave‐Mixing Experiments

Vibrational coherence dynamics in the all-trans retinal chromophore in Bacteriorhodopsin (BR) are investigated by means of temporally and spectrally resolved degenerate four-wave-mixing experiments. The coherence dynamics depend on the excitation wavelength when BR samples are excited at different wavelengths in a spectral range between 520 nm to above 620 nm. The trends in the dynamics observed by tuning of the excitation wavelength allow an assignment of the wave packet dynamics to ground- and excited-state potential energy surfaces. Specifically, the intensity of so-called "out-of-plane" modes of polyene-chain substituents increases for excitation wavelengths near 500 nm. It is shown that this is consistent with the assignment of out-of-plane modes to excited-state coherence dynamics. Moreover, intense low-frequency coherence dynamics around 200 cm(-1) are observed for signal detection in two different spectral regions of excited-state absorption. These modulations are assigned to excited-state dynamics due to the observed dependence on the excitation wavelength. In addition, we show that generally high-frequency modes (>1010 cm(-1)) originate from wave packet motion in the electronic ground state of all-trans retinal.

The Infrared Spectra of the Retinal Chromophore in Bacteriorhodopsin Calculated by a DFT/MM Approach

In the preceding paper (DOI 10.1021/jp902428x), we have derived the polarized force field "PBR" for bacteriorhodopsin (BR) using hybrid methods which combine density functional theory (DFT) with molecular mechanics (MM) models. This polarized force field has enabled extended molecular dynamics (MD) simulations of BR's chromophore binding pocket which closely preserve the experimentally well-known structure. Here, we employ the PBR-MD trajectories obtained for the conformational substates prevalent at physiological temperatures as material for the DFT/MM computation of the chromophore's vibrational spectra. By comparison with DFT results on the structure and vibrational spectra of an isolated chromophore, we identify the structural and spectral changes induced by the protein environment. Comparisons with the wealth of experimental data available in the literature on the chromophore's vibrational spectra yield estimates on the accuracy of the DFT/MM descriptions. We discuss why highly accurate DFT/MM descriptions are expected to become a decisive tool for solving the long-standing enigma of how the light-driven proton pump BR actually works.

Site-Specific Difference 2D-IR Spectroscopy of Bacteriorhodopsin

We demonstrate the extension of the principle of difference Fourier transform infrared (FTIR) spectroscopy to difference 2D-IR spectroscopy. To this end, we measure difference 2D-IR spectra of the protein bacteriorhodopsin in its early J- and K-intermediates. By comparing with the static 2D-IR spectrum of the protonated Schiff base of all-trans retinal, we demonstrate that the 2D-IR spectrum of the all-trans retinal chromophore in bacteriorhodopsin can be measured with the background from the remainder of the protein completely suppressed. We discuss several models to interpret the detailed line shape of the difference 2D-IR spectrum.

Rapid Thermal Tuning of Chromophore Structure in Membrane Protein

We show that the configuration and the optical property of the retinal chromophore in bacteriorhodopsin (bR) can be tuned dynamically from the all-trans configuration to the 13-cis by using a nanosecond laser-induced temperature-jump. The rapid bleach in the visible absorption optical density of retinal has an apparent formation time of ca. 170 ns, whereas the relaxation process finishes within tens of ms. The dynamical transition of retinal from the all-trans to 13-cis species is believed to occur as a result of rapid protein conformational change especially in the vicinity of retinal binding site. Our study reveals the intrinsic dynamical aspect of the retinal chromophore with respect to the protein structure.

Sub-5-fs Real-time Spectroscopy of Transition States in Bacteriorhodopsin During Retinal Isomerization†

By using a sub-5-fs visible laser pulse, we have made the first observation of the vibrational spectra of the transition state during trans-cis isomerization in the retinal chromophore of bacteriorhodopsin (bR(S68). No instant isomerization of the retinal occurs in spite of electron promotion from the bonding pi-orbital to the anti-bonding pi*-orbital. The difference between the in-plane and out-of-plane vibrational frequencies (about 1150-1250 and 900-1000 cm(-1), respectively) is reduced during the first time period. The vibrational spectra after this period became very broad and weak and are ascribed to a "silent state." The silent state lasts for 700-900 fs until the chromophore isomerizes to the cis-C13 = C14 conformation. The frequency of the C = C stretching mode was modulated by the torsion mode of the C13 = C14 double bond with a period of 200 fs. The modulation was clearly observed for four to five periods. Using the empirical equation for the relation between bond length and stretching frequency, we determined the transitional C = C bond length with about 0.01 angstroms accuracy during the torsion motion around the double bond with 1-fs time resolution.

Dynamics of Primary Events in the Photocycle of Excited Bacteriorhodopsin

Transient dynamic behavior of the excited bacteriorhodopsin (BR), which was isolated from the strain H. salinarum, was studied at excitation wavelength from 585 to 639 nm. With the one-color femtosecond (fs) pump-probe technique, we revealed the primary events in BR's photocycle that took place in an ultrafast time scale. From the analysis of the decay components of the dynamical traces, it was evident that the isomerization of the retinal chromophore in BR and the intermediate J's formation occurred within 500 fs. BR exhibited pH-dependent dynamical behaviors. When the medium pH was between 5 and 9, the BR ultrafast dynamics has no obvious change. In contrast, the dynamical curves were obviously affected when the pH was out of that region.

Effect of β-particles on the retinal chromophore in bacteriorhodopsin of Halobacterium salinarium

Bacteriorhodopsin (bR) is an attractive intelligent material. Understanding the mechanism of its light-driven proton pumping outward the cell implicates it in many technical applications, particularly, in what is called optical computers, and the biotechnology is waiting for this promised biological molecule. An ionizing radiation source handling could be computerized in radiation fields. The computer containing such biological material will not be out of reach of the fields of ionizing radiation. So it is interesting to report on the working of such biological computer if it is subjected to ionizing radiation. The functional unit in this molecule is retinal chromophore. In the present work, it is interested to assess the functionality of bR through determining the electronic transition dipole moment of its chromophore. Significant changes in the values of the absorption transition dipole moment were noticed at different doses of β-particles in the range of 0.1– 0.3 kGy . Ionizing radiation-induced changes in bR were followed by intrinsic fluorescence spectroscopy. An analysis of the fluorescence data bears on the tertiary structure of bR. The emission spectrum is, however, red shifted with an increase in intensity with the different doses; in the meanwhile, gradual decrease in the visible absorbance has occurred till almost complete loss is attained. This bleaching due to ionizing radiation may offer an alternative way of data processing in such optical devices based on bR. Nevertheless, bR has proofed to be used as a biological indicator of ionizing radiation. However, the potential of bR for use as a biosensor to detect ionizing radiation should be considered.

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.

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.

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.

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.

Ab Initio Study of 13C NMR Chemical Shifts for the Chromophores of Rhodopsin and Bacteriorhodopsin. 2. Comprehensive Analysis of the 13C Chemical Shifts of Protonated all-trans-Retinylidene Schiff Base

Theoretical analysis was performed for the 13C chemical shifts of the retinal chromophore in bacteriorhodopsin (bR) by means of ab initio NMR shielding calculation, based on the localized orbital/l...

On modeling the vibrational spectra of 14- s-cis retinal conformers in bacteriorhodopsin

The vibrational properties of 13- cis,14- s-trans and 13- cis,14- s-cis protonated retinal Schiff base model compounds are explored with MNDO calculations. In particular, the effect of isomerization about the C 14C 15 single bond on the vibrational properties of the deuterium in-plane rocking vibrations has been examined. Our MNDO calculations, using a variety of lysine models, lysine conformations and Schiff base charge environments, demonstrate that the C 14-D and C 15-D in-plane rocking vibrations in the 14,15-dideuterio retinal protonated Schiff base are strongly coupled in 13- cis,14- s-cis molecules producing a splitting of ca. 80 cm −1 between the symmetric and antisymmetric rocking mode combinations but that these modes are only weakly coupled in 14- s-trans molecules. This analysis demonstrates that the 14,15-dideuterio labeling method developed earlier for determining C 14C 15 conformation (S.P.A. Fodor, W.T. Pollard, R. Gebhard, E.M.M. van den Berg, J. Lugtenburg and R.A. Mathies, Proc. Natl. Acad. Sci. USA, 85 (1988) 2156–2160) is valid, and hence that the structure of the retinal chromophore in bacteriorhodopsin's L 550 intermediate is 13- cis,14- s-trans. The reasons for the misleading conclusions derived from MNDO calculations performed earlier by Schulten and Tavan are discussed.

Long-range effects on the retinal chromophore of bacteriorhodopsin caused by surface carboxyl group modification.

Carboxyl groups of bacteriorhodopsin (bR) that are modified by 1-ethyl-3-[3-(trimethylamino)-propyl]carbodiimide (ETC) have been identified. Reaction of deionized purple membrane with a 400-fold molar excess of ETC or [14C]ETC for 1 h at 0 degree C incorporates about 3.5 mol of ETC/mol of bR. Proteinase K cleavage of ETC-modified bacterioopsin (bO) produced small 14C-labeled peptides. Amino acid sequence analysis showed three major ETC-modified residues: Glu 234, Asp 38, and Glu 74. Proteolysis of purple membrane with papain removes the ETC site at Glu 234. Treatment of ETC-modified, papain-cleaved purple membrane with hydroxylamine removes half of the remaining ETC label. Subsequent cleavage with chymotrypsin, followed by amino acid sequence analysis, revealed that most of the remaining label was at Glu 74. bR modified by ETC primarily at Glu 74 displays two alterations in the retinal chromophore, located in the membrane interior at a distance more than 2 nm away from the modified carboxyl group. (1) The acid-induced purple-to-blue transition undergoes a shift in apparent pK from 3.2 to 2.3. (2) The second-order rate constant for chromophore regeneration from bO and retinal is diminished from 3600 to 1700 M-1 s-1 in membrane sheets. Most of the shift in the pK of the purple-to-blue transition can be explained by the quaternary ammonium ion of ETC attached to Glu 74 overlapping the postulated location of the guanidinium group of Arg 82.(ABSTRACT TRUNCATED AT 250 WORDS)