SUBSTITUTION OF RETINAL BY ANALOGUES IN RETINAL PIGMENTS OF HALOBACTERIUM HALOBIUM. CONTRIBUTION OF BACTERIORHODOPSIN AND HALORHODOPSIN TO PHOTOSENSORY ACTIVITY

Abstract— In Halobacrerium hnlobium. retinal is the chromophore of the light-energy converting pigments bacteriorhodopsin (BR) and halorhodopsin (HR) and of the sensory photosystems. PS 370 and PS 565. In both photosystems as well as in BR and HR. retinal was substituted by retinal analogues. Retinal₂ ( 3,4-dehydro-retinal ) . shifts the main sensitivity maximum of PS 370 and of PS 565 by about 1.5 nm to longer wavelengths. The absorption maxima of BR and HR are both shifted in the same direction, but by 37 nm. 13-Ethylretinal and 13-propylretirnal shift the main sensitivity maximum of each sensory photosystem to shorter wavclengths; the absorption maxima of BR and HR are shifted in the same direction but to a smaller extent. Both sensory photosystems are equally active with retinal and with each of the three analogues as the chromophore. After substitution of retinal by the analogues, the action spectra of PS 565 of the BR-containing strain R₁L₃ show a secondary bensitivity peak in addition to the main peak. This secondary peak matches the absorption maximum of the corresponding BR. In the action spectra of the BR-deficient strainET–15 this secondary peak is missing. Action spectra of PS 565 of the BR-deficient strainL–33, which synthesizes increased amounts of HR. with all retinals show a secondary peak which matches the absorption maximum of the corresponding HR.
The results show that the analogues can substitute retinal in both sensory pigments as well as in BR and HR. Moreover, the data support the previous assumption that both BRand HR, although not required for photosensory activity can contribute to photosensing through PS 565.     

14-Fluoro-bacteriorhodopsin gelatin films for dynamic holography recording

The first dynamic holography recording using 14-fluoro-(14-F) bacteriorhodopsin (BR) gelatin films has been achieved. 14-F BR is an artificial BR pigment made by reconstitution of bacterioopsin (native BR without chromophore) with synthetic 14-F retinal. Low-intensity red light from a cw He-Ne laser was used for dynamic holography recording on the 14-F wild type (WT) BR and 14-F D96N mutant BR in gelatin films. There is not true comparing the diffraction efficiency for 14-F D96N BR and 14-F WT gelatin film, unlike the increased diffraction efficiency for D96N BR gelatin film with native chromophore relative to the WT BR gelatin film with native chromophore. Pre-illumination with blue light of the 14-F BR gelatin films significantly increases the diffraction efficiency of both the 14-F WT and the 14-F D96N BR pigments. The sequential application of blue and red laser beams indicates that 14-F BR gelatin films can be useful for optical memory.     

The effect of the chromophoric group modification on the optical properties of retinal proteins

The effects of chromophoric group structures on the functional properties of bacteriorhodopsin (BR) and proteorhodopsin from E. sibiricum (ESRh) were compared. ESRh retinal binding site was found as preserving the similar stereo- and spatial restrictions on the chromophore structure during the retinal protein reconstitution process (except for C25-analog AR8). It was revealed that the structure peculiarities of the chromophore analog molecules affect the optical parameters of ESRh and BR pigment families in similar ways.     

The Importance of Charge Transfer between the Retinal Chromophore and the Protein Environment in Bacteriorhodopsin: A Theoretical Analysis on Reduced and Oxidized Chromophores

Abstract— The importance of charge transfer(CT) between the retinal chromophore and the protein environment in the ground state of bacteriorhodopsin(BR) has been verified by using ab initio and semiempirical molecular orbital methods. We hypothesize that the chromophore is stabilized in BR by highest occupied molecular orbital-lowest unoccupied molecular orbital(HOMO-LUMO) interaction with the protein environment. If sufficient charge is transferred between two sites due to the strong HOMO-LUMO interaction, the chromophore might be treated as a one-electron reduced species(when it behaves as an electron acceptor), or as a one-electron oxidized one (when it acts as an electron donor).In both optimized geometries, the -conjugated systems exhibit a drastic decrease in bond alternation. To estimate the rotational barrier for thermal isomerization between the al-trans and the 13,15-dicis form, the potential energy curve around these two bonds was computed. The first -_* transition energy was also calculated for an inspection of the opsin shift. The barrier height and the transition energy became much lower as a result of the chromophore reduction. The site selectivity in photo- and thermal isomerization and the opsin shift in BR can be well explained by considering CT from the protein environment to the chromophore.     

ALKALINE QUENCHING OF BACTERIORHODOPSIN TRYPTOPHANYL FLUORESCENCE: EVIDENCE FOR AQUEOUS ACCESSIBILITY OR A HYDROGEN-BONDED CHAIN

Abstract— Comparison between Trp fluorescence yields of membrane-bound bacteriorhodopsin (BR) and retinylidene-free bacterioopsin (BO) is consistent with a model in which all eight Trp residues are active fiuorophores in the latter, while the emission of all but two residues in the former is lost by energy transfer to retinal. The visible chromophore of BR is progressively bleached with increasing pH. Up to pH 12 this bleaching is reversed on reneutralization; but above this the change is irreversible with the appearance of a new absorption band characteristic of free retinal. Emission yields for both proteins decrease with increasingly alkaline pH in a manner typical of energy transfer to weakly-fluorescent tyrosinate. The limiting yields, reached at a pH corresponding to that producing irreversible bleaching of the visible chromophore, agree with an integral value of one remaining active Trp fluorophore in BR and between one and two in BO and show that the bulk of Trp are within the 11 Å Förster energy-transfer distance of Tyr accessible to OH⁻. Current models of the native protein structure of BR arrange the polypeptide chain primarily in a bundle of seven helical segments with axes perpendicular to the lipid bilayer plane and with buried polar residues, including Trp and Tyr, located at intrahelical surfaces. An interpretation of the observed accessibility of buried Tyr to OH⁻ is that an aqueous region exists within the protein structure. Moreover, this putative aqueous region must be close to the retinylidene chromophore and thus may be associated with the light-driven ion transport system. The results are also compatible with energy transfer to internal Tyr residues which are connected via a chain of phenolate hydrogen bonds to a surface Tyr.     

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.     

Analog Retinal Redshifts Visible Absorption of QuasAr Transmembrane Voltage Sensors into Near-infrared

Opsin-based transmembrane voltage sensors (OTVSs) are increasingly important tools for neuroscience enabling neural function in complex brain circuits to be explored in live, behaving animals. However, the visible wavelengths required for fluorescence excitation of the current generation of OTVSs limit optogenetic imaging in the brain to depths of only a few mm due to the strong absorption and scattering of visible light by biological tissues. We report that substitution of the native A1 retinal chromophore of the widely used QuasAr1/2 OTVSs with the retinal analog MMAR containing a methylamino-modified dimethylphenyl ring results in over a 100-nm redshift of the maxima of the absorption and fluorescence emission bands to near 700 and 840 nm, respectively. FT-Raman spectroscopy reveals that at pH 7 QuasAr1 with both the A1 and MMAR chromophores possess predominantly an all-trans protonated Schiff base configuration with the MMAR chromophore exhibiting increased torsion of the polyene single-/double-bond system similar to the O-intermediate of the BR photocycle. In contrast, the A1 and the MMAR chromophores of QuasAr2 exist partially in a 13-cis PSB configuration. These results demonstrate that QuasArs containing the MMAR chromophore are attractive candidates for use as NIR-OTVSs, especially for applications such as deep brain imaging.     

Hydrogen exchange mass spectrometry of bacteriorhodopsin reveals light-induced changes in the structural dynamics of a biomolecular machine

Many proteins act as molecular machines that are fuelled by a nonthermal energy source. Examples include transmembrane pumps and stator-rotor complexes. These systems undergo cyclic motions (CMs) that are being driven along a well-defined conformational trajectory. Superimposed on these CMs are thermal fluctuations (TFs) that are coupled to stochastic motions of the solvent. Here we explore whether the TFs of a molecular machine are affected by the occurrence of CMs. Bacteriorhodopsin (BR) is a light-driven proton pump that serves as a model system in this study. The function of BR is based on a photocycle that involves trans/cis isomerization of a retinal chromophore, as well as motions of transmembrane helices. Hydrogen/deuterium exchange (HDX) mass spectrometry was used to monitor the TFs of BR, focusing on the monomeric form of the protein. Comparative HDX studies were conducted under illumination and in the dark. The HDX kinetics of BR are dramatically accelerated in the presence of light. The isotope exchange rates and the number of backbone amides involved in EX2 opening transitions increase roughly 2-fold upon illumination. In contrast, light/dark control experiments on retinal-free protein produced no discernible differences. It can be concluded that the extent of TFs in BR strongly depends on photon-driven CMs. The light-induced differences in HDX behavior are ascribed to protein destabilization. Specifically, the thermodynamic stability of the dark-adapted protein is estimated to be 5.5 kJ mol(-1) under the conditions of our work. This value represents the free energy difference between the folded state F and a significantly unfolded conformer U. Illumination reduces the stability of F by 2.2 kJ mol(-1). Mechanical agitation caused by isomerization of the chromophore is transferred to the surrounding protein scaffold, and subsequently, the energy dissipates into the solvent. Light-induced retinal motions therefore act analogously to an internal heat source that promotes the occurrence of TFs. Overall, our data highlight the potential of HDX methods for probing the structural dynamics of molecular machines under "engine on" and "engine off" conditions.     

Curing and properties of chloroprene and butadiene rubber (CR/BR) blends cross-linked with copper(I) oxide or copper(II) oxide

This paper discusses the curing and properties of chloroprene and butadiene rubber (CR/BR) blends cross-linked with copper(I) oxide (Cu₂O) or copper(II) oxide (CuO). The results revealed that the cross-linking degree of CR/BR blends decreased with the increasing amount of butadiene rubber (BR) in the blends. The mechanical properties of cured CR/BR blends depended on the proportion of elastomers in blends, as well as on the type and amount of the cross-linking agent (Cu₂O, CuO). The cross-linking of CR/BR/Cu₂O or CR/BR/CuO blends followed according to cationic mechanism, using Lewis acid, copper(I) chloride (CuCl) or copper(II) chloride (CuCl₂) generated in situ. Additionally, the prepared compositions, both unfilled and filled, were characterized by very high resistance to flame.     

Subpicosecond protein backbone changes detected during the green-absorbing proteorhodopsin primary photoreaction

Recent studies demonstrate that photoactive proteins can react within several picoseconds to photon absorption by their chromophores. Faster subpicosecond protein responses have been suggested to occur in rhodopsin-like proteins where retinal photoisomerization may impulsively drive structural changes in nearby protein groups. Here, we test this possibility by investigating the earliest protein structural changes occurring in proteorhodopsin (PR) using ultrafast transient infrared (TIR) spectroscopy with approximately 200 fs time resolution combined with nonperturbing isotope labeling. PR is a recently discovered microbial rhodopsin similar to bacteriorhodopsin (BR) found in marine proteobacteria and functions as a proton pump. Vibrational bands in the retinal fingerprint (1175-1215 cm(-1)) and ethylenic stretching (1500-1570 cm(-1)) regions characteristic of all-trans to 13-cis chromophore isomerization and formation of a red-shifted photointermediate appear with a 500-700 fs time constant after photoexcitation. Bands characteristic of partial return to the ground state evolve with a 2.0-3.5 ps time constant. In addition, a negative band appears at 1548 cm(-1) with a time constant of 500-700 fs, which on the basis of total-15N and retinal C15D (retinal with a deuterium on carbon 15) isotope labeling is assigned to an amide II peptide backbone mode that shifts to near 1538 cm(-1) concomitantly with chromophore isomerization. Our results demonstrate that one or more peptide backbone groups in PR respond with a time constant of 500-700 fs, almost coincident with the light-driven retinylidene chromophore isomerization. The protein changes we observe on a subpicosecond time scale may be involved in storage of the absorbed photon energy subsequently utilized for proton transport.     

pH-dependent transitions in xanthorhodopsin

Xanthorhodopsin (XR), the light-driven proton pump of the halophilic eubacterium Salinibacter ruber, exhibits substantial homology to bacteriorhodopsin (BR) of archaea and proteorhodopsin (PR) of marine bacteria, but unlike them contains a light-harvesting carotenoid antenna, salinixanthin, as well as retinal. We report here the pH-dependent properties of XR. The pKa of the retinal Schiff base is as high as in BR, i.e. > or =12.4. Deprotonation of the Schiff base and the ensuing alkaline denaturation cause large changes in the absorption bands of the carotenoid antenna, which lose intensity and become broader, making the spectrum similar to that of salinixanthin not bound to XR. A small redshift of the retinal chromophore band and increase of its extinction, as well as the pH-dependent amplitude of the M intermediate indicate that in detergent-solubilized XR the pKa of the Schiff base counterion and proton acceptor is about 6 (compared to 2.6 in BR, and 7.5 in PR). The protonation of the counterion is accompanied by a small blueshift of the carotenoid absorption bands. The pigment is stable in the dark upon acidification to pH 2. At pH < 2 a transition to a blueshifted species absorbing around 440 nm occurs, accompanied by loss of resolution of the carotenoid absorption bands. At pH < 3 illumination of XR with continuous light causes accumulation of long-lived photoproduct(s) with an absorption maximum around 400 nm. The photocycle of XR was examined between pH 4 and 10 in solubilized samples. The pH dependence of recovery of the initial state slows at both acid and alkaline pH, with pKas of 6.0 and 9.3. The decrease in the rates with pKa 6.0 is apparently caused by protonation of the counterion and proton acceptor, and that at high pH reflects the pKa of the internal proton donor, Glu94, at the times in the photocycle when this group equilibrates with the bulk.     

ISOMERIZATION OF THE RETINYLIDENE CHROMOPHORE OF BACTERIORHODOPSIN IN LIGHT ADAPTATION: INTRINSIC ISOMERIZATION OF THE CHROMOPHORE AND ITS CONTROL BY THE APO-PROTEIN

Abstract— The dependence of the isomeric configuration of the retinylidene chromophore of bacteriorhodopsin on the pH value and on the wavelength of irradiation (in a photostationary state) were examined by high performance liquid chromatographic analyses of extracted retinal. The process of isomerization of the chromophore during light adaptation was also traced. More than 93% of all- trans and less than 5% of 13- cis retinal were extracted in the photostationary state for irradiation at 560 nm in the pH region of5–9 as well as for irradiation in the wavelength region of 400–650 nm at pH 7. Comparison of the above photostationary state composition with that of protonated n -butylamine Schiff base of retinal indicates that strong constraint is applied to the chromophore by the apo-protein. The constraint can be changed at low or high pH by a partial denaturation or transition of the apo-protein, which results in the generation of 11- cis retinal in the extract. At higher photon density, the isomerization process of the chromophore during light adaptation at pH 7 was characterized, as extracted isomeric retinal, by (1) the initial decrease in 13- cis and increase in all- trans , (2) a subsequent, transient toward the above photostationary state composition. The results are discussed in terms of both the photoisomerization pattern inherent in the retinylidene chromophore and the control by the apo-protein.     

LIGHT INDUCED ISOMERISATION, AT ACIDIC pH, INITIATES HYDROLYSIS OF BACTERIORHODOPSIN TO BACTERIO-OPSIN AND 9-CIS-RETINAL

Abstract— Bacteriorhodopsin (BR) from the purple membrane of Haiobacterium halobium contains covalently bound retinal in the 13- cis and all- trans configurations. Several forms of bacteriorhodopsin are known, with different absorption maxima which are designated as BRλ_max (nm). At acidic pH, BR605 is formed from BR560. The following sequence of reactions was found, which is initiated by irradiation of BR605 with red light:

An all- trans /13- cis to 9- cis isomerisation occurs in the light induced reaction BR605 ∼ BR500. BR500 seems to contain covalently bound retinal, whereas BR390 contains free retinal. By irradiation with light, BR500, BR450 and BR390 can be reconverted to BR560.     

PRIMARY PHOTOCHEMISTRY OF BACTERIORHODOPSIN: COMPARISON OF FOURIER TRANSFORM INFRARED DIFFERENCE SPECTRA WITH RESONANCE RAMAN SPECTRA

Abstract —Fourier transform infrared (FTIR) difference spectra of the BR→rK transition in bacteriorhodopsin at 77→K are compared with analogous resonance Raman difference spectra obtained using a spinning sample cell at 77→K. The vibrational frequencies observed in the FTIR spectra of native purple membrane and of purple membrane regenerated with 15-deuterioretinal are in good agreement with the frequencies observed in the Raman spectra, indicating that the lines in the FTIR difference spectra arise predominantly from retinal chromophore vibrations. This agreement confirms that the spinning cell method for obtaining resonance Raman spectra of K minimizes potential contributions from unwanted photoproducts. The unexpected similarity between the resonance Raman scattering intensities and the FTIR absorption intensities for BR and K is discussed in terms of the delocalized electronic structure of the chromophore. Finally, comparison of the Schiff base regions of the K Raman and FTIR spectra provide additional information on the assignment of its Schiff base vibration.     

Femtosecond primary events in bacteriorhodopsin and its retinal modified analogs: revision of commonly accepted interpretation of electronic spectra of transient intermediates in the bacteriorhodopsin photocycle

Femtosecond primary events in bacteriorhodopsin (BR) and its retinal modified analogs are discussed. Ultrafast time resolved electronic spectra of the primary intermediates induced in the BR photocycle are discussed along with spectral and kinetic inconsistencies of the previous models proposed in the literature. The theoretical model proposed in this paper based on vibrational coupling between the electronic transition of the chromophore and intramolecular vibrational modes allows us to calculate the equilibrium electronic absorption band shape and the hole burning profiles. The model is able to rationalize the complex pattern of behavior for the primary events in BR and explain the origin of the apparent inconsistencies between the experiment and the previous theoretical models. The model presented in the paper is based on the anharmonic coupling assumption in the adiabatic approximation using the canonical transformation method for diagonalization of the vibrational Hamiltonian instead of the commonly used perturbation theory. The electronic transition occurs between the Born-Oppenheimer potential energy surfaces with the electron involved in the transition being coupled to the intramolecular vibrational modes of the molecule (chromophore). The relaxation of the excited state occurs by indirect damping (dephasing) mechanisms. The indirect dephasing is governed by the time evolution of the anharmonic coupling constant driven by the resonance energy exchange between the intramolecular vibrational mode and the bath. The coupling with the intramolecular vibrational modes results in the Franck-Condon progression of bands that are broadened due to the vibrational dephasing mechanisms. The electronic absorption line shape has been calculated based on the linear response theory whereas the third order nonlinear response functions have been used to analyze the hole burning profiles obtained from the pump-probe time-resolved measurements. The theoretical treatment proposed in this paper provides a basis for a substantial revision of the commonly accepted interpretation of the primary events in the BR photocycle that exists in the literature.     

Role of endogenous lipids in the chromophore regeneration of bacteriorhodopsin

The regeneration method of Khorana [J. Biol. Chem. 262 (1987) 9271] has been modified in order to study the effect of endogenous archaeabacterial lipids and, in particular, of glycocardiolipin (GlyC) in the refolding and chromophore regeneration of bacteriorhodopsin (BR). BR refolding and chromophore regeneration could be obtained in the presence of endogenous lipid mixtures containing or not containing glycocardiolipin; however, the kinetics of bacteriorhodopsin regeneration in the presence of glycocardiolipin was faster than in its absence. These results show for the first time that the interaction of glycocardiolipin with bacteriorhodopsin favours its refolding from the denaturated state and the chromophore regeneration.     

RESONANCE RAMAN SPECTRA OF BACTERIORHODOPSIN MUTANTS WITH SUBSTITUTIONS AT ASP-85, ASP-96, AND ARG-82

Detergent solubilized bacteriorhodopsin (BR) proteins which contain alterations made by site-directed mutagenesis (Asp-96----Asn, D96N; Asp-85----Asn, D85N; and Arg-82----Gln, R82Q) have been studied with resonance Raman spectroscopy. Raman spectra of the light-adapted (BRLA) and M species in D96N are identical to those of native BR, indicating that this residue is not located near the chromophore. The BRLA states of D85N and especially R82Q contain more of the 13-cis, C = N syn (BR555) species under ambient illumination compared to solubilized native BR. Replacement of Asp-85 with Asn causes a 25 nm red-shift of the absorption maximum and a frequency decrease in both the ethylenic (-7 cm-1) and the Schiff base C = NH+ (-3 cm-1) stretching modes of BRLA. These changes indicate that Asp-85 is located close to the protonated retinal Schiff base. The BRLA spectrum of R82Q exhibits a slight perturbation of the C = NH+ band, but its M spectrum is unperturbed. The Raman spectra and the absorption properties of D85N and R82Q suggest that the protein counterion environment involves the residues Asp-85-, Arg-82+ and presumably Asp-212-. These data are consistent with a model where the strength of the protein-chromophore interaction and hence the absorption maximum depends on the overall charge of the Schiff base counterion environment.     

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.     

Photochromism of Anabaena sensory rhodopsin

Protein-controlled photochemical reactions often mediate biological light-signal and light-energy conversions. Microbial rhodopsins possess all-trans or 13-cis retinal as the chromophore in the dark, and in the light-driven proton pump, bacteriorhodopsin (BR), the stable photoproduct at the end of the functional cycle of the all-trans form is 100% all-trans. In contrast, a microbial rhodopsin discovered in Anabaena PCC7120 is believed to function as a photochromic sensor. For Anabaena sensory rhodopsin (ASR), the photoreaction is expected to be not cyclic, but photochromic. The present low-temperature UV-visible spectroscopy of ASR indeed revealed that the stable photoproduct of the all-trans form in ASR is 100% 13-cis, and that of the 13-cis form is 100% all-trans. The complete photocycle for the proton pump in BR and the complete photochromism for the chromatic sensor of ASR are highly advantageous for their functions. Thus, the microbial rhodopsins have acquired unique photoreactions, in spite of their similar structures, during evolution.     

The laser-induced blue state of bacteriorhodopsin: mechanistic and color regulatory roles of protein-protein interactions, protein-lipid interactions, and metal ions

In this paper we characterize the mechanistic roles of the crystalline purple membrane (PM) lattice, the earliest bacteriorhodopsin (BR) photocycle intermediates, and divalent cations in the conversion of PM to laser-induced blue membrane (LIBM; lambda(max)= 605 nm) upon irradiation with intense 532 nm pulses by contrasting the photoconversion of PM with that of monomeric BR solubilized in reduced Triton X-100 detergent. Monomeric BR forms a previously unreported colorless monomer photoproduct which lacks a chromophore band in the visible region but manifests a new band centered near 360 nm similar to the 360 nm band in LIBM. The 360 nm band in both LIBM and colorless monomer originates from a Schiff base-reduced retinyl chromophore which remains covalently linked to bacterioopsin. Both the PM-->LIBM and monomer-->colorless monomer photoconversions are mediated by similar biphotonic mechanisms, indicating that the photochemistry is localized within single BR monomers and is not influenced by BR-BR interactions. The excessively large two-photon absorptivities (> or =10(6) cm(4) s molecule(-1) photon(-1)) of these photoconversions, the temporal and spectral characteristics of pulses which generate LIBM in high yield, and an action spectrum for the PM-->LIBM photoconversion all indicate that the PM-->LIBM and Mon-->CMon photoconversions are both mediated by a sequential biphotonic mechanism in which is the intermediate which absorbs the second photon. The purple-->blue color change results from subsequent conformational perturbations of the PM lattice which induce the removal of Ca(2+) and Mg(2+) ions from the PM surface.