Studies on pyrylretinal analogues of bacteriorhodopsin

The retinal analogues 3-methyl-5-(1-pyryl)-2E,4E-pentadienal (1) and 3,7-dimethyl-9-(1-pyryl)-2E,4E,6E,8E-nonatetr aenal (2), which contain the tetra aromatic pyryl system, have been synthesized and characterized in order to examine the effect of the extended ring system on the binding capabilities and the function of bacteriorhodopsin (bR). The two bR mutants, E194Q and E204Q, known to have distinct proton-pumping patterns, were also examined so that the effect of the bulky ring system on the proton-pumping mechanism could be studied. Both retinals formed pigments with all three bacterioopsins, and these pigments were found to have absorption maxima in the range 498-516 nm. All the analogue pigments showed activity as proton pumps. The pigment formed from wild-type apoprotein bR with 1 (with the shortened polyene side chain) showed an M intermediate at 400 nm and exhibited fast proton release followed by proton uptake. Extending the polyene side chain to the length identical with retinal, analogue 2 with wild-type apoprotein gave a pigment that shows M and O intermediates at 435 nm and 650 nm, respectively. This pigment shows both fast and slow proton release at pH 7, suggesting that the pKa of the proton release group (in the M-state) is higher in this pigment compared to native bR. Hydrogen azide ions were found to accelerate the rise and decay of the O intermediate at neutral pH in pyryl 2 pigment. The pigments formed between 2 and E194Q and E204Q showed proton-pumping behavior similar to pigments formed with the native retinal, suggesting that the size of the chromophore ring does not alter the protein conformation at these sites.     

AZIDOTETRAFLUOROPHENYL RETINAL ANALOGUE: SYNTHESIS AND BACTERIORHODOPSIN PIGMENT FORMATION

Abstract —The retinal derivative, all-truns-9–(4-azido-2,3,5,6-tetrafluorophenyl)-3,7-dimethyl-2,4,6,8-nonatetraenal, was synthesized by two routes as a potential photoactivatable cross-linking agent for studies in bacteriorhodopsin (BR) of the chromophore interaction with its apoprotein. The retinal analogue formed a stable, moderately functional BR pigment confirming that the ring cavity of the retinal binding site has a significant tolerance for derivatization on that portion of the molecule. Attempts to cross-link the azido chromophore to the protein by photoactivation were unsuccessful. The electron delocalization effect of the conjugated polyene side chain of the retinal appears to interfere with the formation or reactivity of the nitrene intermediate to the extent that photoactivated cross-linking is not achieved. These results demonstrate a limitation to the use of fluorinated aryl azides as photoaffinity reagents.     

Ring oxidized retinals form unusual bacteriorhodopsin analogue pigments.

Three ring oxidized retinal analogues have been isolated from the exhaustive oxidation of all-trans retinal. All-trans 4-oxoretinal and 2,3-dehydro-4-oxoretinal have similar absorption maxima to that of all-trans retinal and have been shown to be in the 6-s-cis conformation in solution. Pigments formed with bacterioopsin exhibit absorption maxima (520 nm) blue-shifted from that of bacteriorhodopsin (bR), indicating a disturbance of the external point charge by the electronegative carbonyl moiety at the 4 position. The third analogue contains a ring contracted to a cyclopentenyl-alpha,beta-dione. Unlike the majority of retinals, this analogue displays a 6-s-trans conformation in solution and has a red-shifted absorption maximum at 435 nm. The resulting bR analogue pigment (515 nm) is formed five times faster than the other oxoretinal pigments. All three oxoretinal pigments show an irreversible 20 nm blue shift upon exposure to white light. The 4-oxo and 2,3-dehydro-4-oxoretinal pigments, after irradiation, undergo a small reversible blue shift (4-8 nm) on dark adaptation. These two pigments pump protons, although with slowed photocycle kinetics, demonstrating that these structural changes (addition of the carbonyl at the C-4 and insertion of a double bond in the ring) do not block the function of the pigment. Extraction of the C-15 tritiated analogue retinals from illuminated and non-illuminated pigments of all three oxoretinals yield identical results. Therefore, any crosslinking of these oxoretinals to the protein is by linkages which are unstable to the extraction procedures.     

PHOTOCHEMICAL STUDIES OF ARTIFICIAL BACTERIORHODOPSINS

Abstract— Three artificial bacteriorhodopsins are prepared [from synthetic aromatic and bicyclic analogues of retinal and exposed to spectroscopic and pulsed lader photolysis studies. The spectra of the pigments, all perturbed in the ring region of the molecule, are markedly blue shifted in respect to natural bacteriorhodopsin. The shift is attributed to a decreased effect of a protein charge in the vicinity of the ring, in agreement with the point-charge model of Nakanishi et al. , 1980. The photocycles of the synthetic pigments exhibit a primary red-shifted (K) intermediate and a blue shifted (M) transient, analogous to those observed for the natural pigment. Such observations impose considerable limitations, both on the possible chromophore conformational changes and on the effects of neighbouring protein charges associated with the photocycle.
It is concluded that only the Schiff base counter-ion, but not the ring charge, may be associated with the generation of the primary red shifted K species. Moreover, the rigidity imposed on the polyene by the additional ring in the bicyclic analogue shows that the photocycle can not be initiated by conformational changes in the retinyl moiety up to the C₉ carbon in the polyene chain. It is also observed that the K→L process in the photocycle is considerably slower in the case of the synthetic pigments. The observation is rationalized by attributing the process to a conformational change in the polyene moiety catalyzed by the ring protein charge.     

THE 'OPSIN SHIFT' IN BACTERIORHODOPSIN: STUDIES WITH ARTIFICIAL BACTERIORHODOPSINS

Abstract— The difference (in cm⁻¹) in absorption maxima between the protonated Schiff base of retinals and the pigment derived therefrom has been defined as the opsin shift. It represents the influence of the opsin binding site on the chromophore. The analysis of the opsin shifts of a series of dihydrobacteriorhodopsins has led to the external point-charge model, which in addition to a counter anion near the Schiff base ammonium, carries another negative charge in the vicinity of the β-ionone ring. This is in striking contrast to the external point-charge model proposed earlier for the bovine visual pigment. The absorption maxima of rhodopsins formed from bromo- and phenyl retinals support the two models. A retinal carrying a photoaffinity label has yielded a nonbleachable bacteriorhodopsin.     

A NOVEL BACTERIORHODOPSIN ANALOGUE WITH CONFORMATIONALLY 6-s-cis FIXED RETINALS

Abstract— The artificial pigments of bacteriorhodopsin (bR) were synthesized with retinal analogues which have 6-s-cis fixed conformation. In order to study the shape of binding pocket around β-ionone ring of retinal, we used three different 6-s-cis fixed retinals (1–3), which have a gem-dimethyl group at a different position of the ring structure. These three 6-s-cis fixed retinals produced bR analogue pigments with absorption maxima at 574, 594 and 584 nm, respectively. These analogue pigments showed proton pumping activities upon incorporation into vesicles, indicating that 6-s-trans structure of retinal is not necessary to pump protons. Although these retinal analogues possess the same conjugated π-electron systems, the wavelengths of maximum absorbance of the analogue pigments were different from each other, indicating a distinctly steric interaction between retinal ring portion and apoprotein. The analogue pigments were also different from each other in regeneration rate and the number of the final products. These differences resulted from a difference in the position of the dimethyl substituent at the ring structure. Based on the results the molecular structure around β-ionone ring of retinal-binding pocket was discussed.     

RESONANCE RAMAN STUDIES OF BACTERIORHODOPSIN ANALOGUES

Abstract— We present the results of resonance Raman measurements on a series of bacteriorhodopsin (bR) analogues formed from synthetic retinals which have replaced the native chromophore in the active site. Specifically, 5,6-dihydro-bR, 13-desmethyl-bR, 10-methyl-bR, 14-methyl-bR, and 10.14-dimethyl-bR have been studied. All five analogues bind and form Schiff base retinal-apoprotein linkages. While the Schiff base linkages of 5,6-dihydro-bR, 13-desmethyl-bR, and 10-methyl-bR are protonated, like the native chromophore, the 14-methyl-bR, and 10,14-dimethyl-bR Schiff bases are unprotonated. These results suggest that the binding site of bacteriorhodopsin near the Schiff base moiety is different from that of rhodopsin. The protonated Schiff base -C=NH- stretching frequency of 5.6-dihydro-bR lies at 1660 cm^-1 which is unusually high for a bacteriorhodopsin based pigment. The downward shift upon deuteration is 16 cm^-1, essentially identical to that measured for bacteriorhodopsin. This and the other analogue results strongly reinforce our previous arguments that the Schiff base stretching frequency is determined in large part by two factors, the C=N force constant and the stretch interaction with C=N-H bend. On the other hand, the deuterium isotope effect is determined primarily by the stretch-bend interaction.     

VISUAL PIGMENTS AND BACTERIORHODOPSINS FORMED FROM AROMATIC RETINAL ANALOGS

Abstract— Rhodopsin (Rh) and bacteriorhodopsin (bR) analogs have been prepared from retinals containing various aromatic and heterocyclic nuclei. In the case of Rh, aromatic methyl substituents facilitate the regeneration and stabilize the pigments formed; in bR, however, methyl substituents seem to have little influence. Rhodopsins derived from unsubstituted aromatic retinals show fine structure in the relatively stable "pre-pigment" intermediate and their maxima are red-shifted compared to pigments derived from methylated aromatic retinals. This implies that in these aromatic rhodopsins the ring moiety adopts a more planar conformation when unsubstituted. In bR derivatives also the aromatic ring adopts a close-to-planar conformation when unsubstituted, but comparison with indene-derived bR suggests that even the unsubstituted phenyl ring may not be coplanar with the side-chain.     

ISOMER COMPOSITION and SPECTRA OF THE DARK and LIGHT ADAPTED FORMS OF ARTIFICIAL BACTERIORHODOPSINS*

Abstract– The isomer composition and spectral properties of 15 artificial bacteriorhodopsin (bR) pigments, based on a series of retinal analogs with polyene residue modified below C₉ are determined for both dark-adapted (DA) and light-adapted (LA) forms. Similarly to native bR, in all cases only two isomers, C₁₃=C₁₄cis (13-cis) and M-trans, are observed. However, the artificial DA pigments have a lower 13-d.s content than native DA bR (? 66%) while the corresponding LA pigments have a much higher 13-cis content (11-69%) than native LA bR (<2%). Thus, in variance with the native pigment, in all of the artificial systems light also induced the reversed all-trans13-cis process. The data are accounted for in terms of specific steric interactions between the polyene and the protein binding site which allow a (C₁₅-anti)(C_ls-syn) isomerization during the photocycle of the artificial pigments, but not in the case of native bR. This accounts for the high proton pumping efficiency of the natural pigment. The nature of a highly red shifted light-adapted form of two of the artificial pigments is investigated and discussed. It is also shown that, in variance with native bR, several artificial pigments exhibit identical absorption spectra for their 13-cis and all-trans isomers. It is concluded that the spectral data for the above species of artificial pigments do not lead to a clear molecular model for the origin of the spectral shift between 13-cis and all-trans bR.     

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.     

CONFORMATIONAL ANALYSIS OF THE RHODOPSIN CHROMOPHORE USING BICYCLIC RETINAL ANALOGUES

Abstract— For investigation of the chromophore conformation around the trimethyl cyclohexene ring and of the origin of the induced β-circular dichroism band in rhodopsin, two C₆-C₇ single bond-fixed retinal analogues, 6s-cb- and 6s-trans-locked bicyclic retinals (6 and 7, respectively) were synthesized and incorporated into bovine opsin in CHAPS-PC mixture. 6s-cb- and 6s-tram-Locked rhodopsin analogues (8 and 9 ) with A max at 539 and 545 nm, respectively, were formed. Interestingly, both 8 and 9 displayed α- and β-circular dichroism bands. The ellipticity of α-bands are similar in each other, while the β-band of 8 was about three times stronger than that of 9. Irradiation of 6s-trans-locked rhodopsin, 9, in the presence of hyroxylamine, resulted in the formation of only one of the enantiomers of 6s-rrans-locked retinal oxime showing a positive circular dichroism signal at around 390 nm. This fact strongly suggests that the retinal binding site of rhodopsin shows a chiral discrimination. From these experimental results, the interactions between the trimethyl cyclohexene ring portion in the chromophore and the neighbouring protein moiety in the rhodopsin molecule are discussed.     

TIME-RESOLVED FLUOROMETRY OF BACTERIORHODOPSIN

Abstract A mode-locked Nd:YAG laser was used to excite the aromatic amino acid residues of bacteriorhodopsin in the purple membrane and the tryptophan (Trp) fluorescence decay analyzed with a streak camera (λ> 380 nm). The decay kinetics are resolvable into two first-order half-times (1.5 and 0.17 ns, respectively), while for retinylidene-free bacterioopsin, only the longer-lived Trp emission was observed. The shorter-lived species reappeared upon regeneration of bacteriorhodopsin by addition of retinal to bacterioopsin but not on treatment of the latter with an equivalent of retinol. It is proposed that these results are consistent with a structural model in which the 7-8 Trp's distributed among sections A, C, E and F of the seven helical segments A-G of native bacteriorhodopsin are distinguishable by their distances from the chromophore. Assuming a Förster mechanism for energy transfer with R_o= 25 and 32 Å, respectively, for retinylidene chromophore and retinol the Trp's may be divided into two groups: (i) those completely quenched by retinol and partly quenched by retinal (τ= 0.17) with R ≃ 18 Å and (ii) those (τ= 1.5 ns) which are quenched neither by chromophore nor retinol with R > ca. 30 Å. These results are consistent with and support some of the best models of Engelman et al. (1980) for the protein conformation in the purple membrane.     

SHAPE OF THE CHROMOPHOIW BINDING SITE IN pharaonis PHOBORHODOPSIN FROM A STUDY USING RETINAL ANALOGS

Abstract To investigate the shape of the chromophore binding site of pharaonis phoborhodopsin (ppR), ppR-opsin was incubated with five ring-modified retinal analogs: an acyclic retinal, phenylretinal, α-retinal, cyclohexylretinal and 5-isopropyl-α-retinal. The experimental results were compared with those obtained from bacteriorhodopsin-opsin (bR-opsin) and the same retinal analogs. It was suggested that ring chain conformation is important in affecting the spectral shoulder unique for the absorption spectrum of ppR. The rate of pigment formation depended greatly on the analogs used with the planar analogs showing rapid formation. Thus, we concluded that the space of the retinal binding site of ppR is restricted to the plane of the cyclohexenyl ring of the chromophore, whereas that of bR is less restricted.     

Steric barrier to bathorhodopsin decay in 5-demethyl and mesityl analogues of rhodopsin

Absorbance difference spectra were recorded from 20 ns to 1 micros after 20 degrees C photoexcitation of artificial visual pigments derived either from 5-demethylretinal or from a mesityl analogue of retinal. Both pigments produced an early photointermediate similar to bovine bathorhodopsin (Batho). In both cases the Batho analogue decayed to a lumirhodopsin (Lumi) analogue via a blue-shifted intermediate, BSI, which formed an equilibrium with the Batho analogue. The stability of 5-demethyl Batho, even though the C8-hydrogen of the polyene chain cannot interact with a ring C5-methyl group to provide a barrier to Batho decay, raises the possibility that the 5-demethylretinal ring binds oppositely from normal to form a pigment with a 6-s-trans ring-chain conformation. If 6-s-trans binding occurred, the ring C1-methyls could replace the C5-methyl in its interaction with the chain C8-hydrogen to preserve the steric barrier to Batho decay, consistent with the kinetic results. The possibility of 6-s-trans binding for 5-demethylretinal also could account for the unexpected blue shift of 5-demethyl visual pigments and could explain why 5-demethyl artificial pigments regenerate so slowly. Although the mesityl analogue BSI's absorption spectrum was blue-shifted relative to its pigment spectrum, the blue shift was much smaller than for rhodopsin's or 5-demethylisorhodopsin's BSI. This suggests that increased C6-C7 torsion may be responsible for some of BSI's blue shift, which is not the case for mesityl analogue BSI either because of reduced spectral sensitivity to C6-C7 torsion or because the symmetry of the mesityl retinal analogue precludes having 6-s-cis and 6-s-trans conformers. The similarity of the mesityl analogue BSI and native BSI lambda(max) values supports the idea that BSI has a 6-s angle near 90 degrees, a condition which could disconnect the chain (and BSI's spectrum) from the double bond specifics of the ring.     

THE WHITE MEMBRANE OF CRYSTALLINE BACTERIOOPSIN IN HALOBACTERIUM HALOBIUM STRAIN R1mW AND ITS CONVERSION INTO PURPLE MEMBRANE BY EXOGENOUS RETINAL

Abstract— A new strain isolated from Halobacterium halobium designated R₁mW, contained negligible amounts of isoprenoid pigments, had a yellowish white color due to respiratory pigments and showed no proton movement in response to light. However, addition of all-trans-retinal converted R₁mW into purple cells. Formation of both halorhodopsin and bacteriorhodopsin was indicated by induction of light-dependent proton uptake and release, respectively. Both haloopsin and bacterioopsin were thus postulated to be present in R₁mW. Electron micrographs of freeze-fractured cytoplasmic membranes revealed patches in a hexagonal array of trimeric particles, comparable to the purple membrane structure. These white membrane patches were isolated by procedures similar to those for the purple membrane. The white membrane's buoyant density was about 1.18 g/m/, and its main component migrated on sodium dodecylsulfate polyacrylamide gels at the same rate as bacteriorhodopsin. The white membrane showed only a small absorption peak at ～410nm due to contaminating respiratory pigments and a strong absorption at around 275 nm and shorter wavelengths. The white membrane was thus considered to be mainly composed of bacterioopsin, which was readily converted into bacteriorhodopsin by an addition of all-trans-retinal. The absorption and CD spectra of the white membrane were measured before and after addition of retinal. The molar extinction coefficient of dark-adapted bacteriorhodopsin formed was determined to be 53000M^?1 cm^?1 at 560 nm from retinal binding studies. The CD spectrum of the white membrane was negligible in the visible region but showed several bands assigned to aromatic and backbone structures in the UV region. Retinal addition caused considerable changes in the spectrum, yielding the CD spectrum of crystalline purple membrane bacteriorhodopsin. The white membrane thus appears to be a preparation suitable for structure-function studies of bacteriorhodopsin.     

PHOTOSELECTION AND TRANSIENT LINEAR DICHROISM WITH ORIENTED IMMOBILIZED PURPLE MEMBRANES: EVIDENCE FOR MOTION OF THE C(20)-METHYL GROUP OF THE CHROMOPHORE TOWARDS THE CYTOPLASMIC SIDE OF THE MEMBRANE*

Abstract— We have determined the transition dipole moment orientation of the chromophore during the photocycle of bacteriorhodopsin by photoselection and time-resolved linear dichroism experiments with samples of oriented immobilized purple membranes. This technique offers two important advantages over experiments with isotropic aqueous suspensions: (1) the depolarization due to the rotational diffusion of the membranes is eliminated, (2) the sensitivity for detecting the orientation of the transition dipole moment of intermediates is greatly increased. The appropriate equations for the analysis of time-resolved linear dichroism experiments with samples of oriented immobilized membranes will be presented. In the transition from the ground state of bacteriorhodopsin to the M-intermediate, the transition dipole moment tilts out of the plane of the membrane by about 3°. On the basis of current structural information on the plane of the chromophore and the orientation of its C(19) and C(20) methyl groups, a tilt of the transition dipole moment into the plane of the membrane would have been expected if it is assumed that the orientation of the conjugated polyene chain from C(5) to C(13) is the same in both states. The experimental result may be explained by an 11° tilt of the C(5) to C(13) part of the chain out of the plane of the membrane with the C(20) methyl group moving towards the cytoplasmic side of the membrane by about 1.7 Å and the cyclohexene ring staying fixed. This interpretation is supported by recent neutron diffraction experiments on the chromophore position in the M-intermediate.     

HEAVY-ATOM LABELLED RETINAL ANALOGUES LOCATED IN BACTERIORHODOPSIN BY X-RAY DIFFRACTION*

Abstract– The location of the heavy-atom label of three different retinal analogues in the plane of the purple membrane was determined by x-ray diffraction. The three analogues, i.e. 9-bromoretinal and 13-bromoretinal labelled in the polyene chain as well as the ring-labelled HgCI-retinal, were incorporated into bacteriorhodopsin (BR) either biosynthetically using a retinal-deficient mutant strain of Halobacterium halobium or with photochemically bleached bacterioopsin. All BR samples regenerated with retinal analogues were functionally active as proton pumps. The diffraction data show that the cyclohexene ring of retinal is situated in the corner formed by helices 4E and 5D. the 13-methyl group adjacent to helix 6C and therefore the Schiff's base nitrogen about midway between helices 6C and 2G. The 9-bromo label is found slightly off the line connecting the two other labels, directed towards helix 3F, suggesting torsion of the polyene chain or slight incline of the ring. The position and orientation of retinal obtained in our experiments are in agreement with data from neutron diffraction and high-resolution electron microscopy. This indicates that the heavy-atom labeled chromophores, 9-bromo- and 13-bromo- as well as HgCI-retinal, are isomorphously incorporated into BR. These samples will allow kinetic investigation of light-induced structural changes of retinal during BR's pumping cycle using x-ray diffraction as well as extended x-ray absorption fine structure experiments probing changes in the retinal neighbourhood between different intermediaies of the photocycle.     

ANALYZING THE RED-SHIFT CHARACTERISTICS OF AZULENIC,NAPHTHYL, OTHER RING-FUSED AND RETINYL PIGMENT ANALOGS OF BACTERIORHODOPSIN*

Prompted by the near infrared-absorbing properties of some of the azulenic bacteriorhodopsin (bR) analogs, we have analyzed their absorption characteristics along with 11 new related ring-fused analogs and the corresponding Schiff bases (SB) and protonated Schiff bases (PSB). The following three factors are believed to contribute to the total red shift of each of the pigment analogs (α_RS): perturbation of the basic chromophore (SB shift, ΔSB), protonation of the SB (PSB shift, PSBS) and protein perturbation (the opsin shift, OS). For each factor, effects of structural modifications were examined. For the red-shifted pigments, percent OS has been suggested as an alternate way of measuring protein perturbation. Computer-simulated chromophores provided evidence against any explanation involving altered shapes of the binding pocket as a major cause for absorption differences. Implications of the current bR results on preparation of further red-shifted bR and possible application to visual pigment analogs are discussed.     

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.     

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.