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PubMed1994

Met-145 is a key residue in the dark adaptation of bacteriorhodopsin homologs.

Composition of retinal isomers in three proton pumps (bacteriorhodopsin, archaerhodopsin-1, and archaerhodopsin-2) was determined by high performance liquid chromatography in their light-adapted and dark-adapted states. In the light-adapted state, more than 95% of the retinal in all three proton pumps were in the all-trans configuration. In the dark-adapted state, there were only two retinal isomers, all-trans and 13-cis, in the ratio of all-trans: 13-cis = 1:2 for bacteriorhodopsin, 1:1 for archaerhodopsin-1, and 3:1 for archaerhodopsin-2. The difference in the final isomer ratios in the dark-adapted bacteriorhodopsin and archaerhodopsin-2 was ascribed to the methionine-145 in bacteriorhodopsin. This is the only amino acid in the retinal pocket that is substituted by phenylalanine in archaerhodopsin-2. The bacteriorhodopsin point-mutated at this position to phenylalanine dramatically altered the final isomer ratio from 1:2 to 3:1 in the dark-adapted state. This point mutation also caused a 10 nm blue-shift of the adsorption spectrum, which is similar to the shift of archaerhodopsin-2 relative to the spectra of bacteriorhodopsin and archaerhodopsin-1.

Adaptation, Physiological↗
PubMed1999

Bleaching of bacteriorhodopsin by continuous light.

A new two step photobleaching process is observed under continuous illumination of bacteriorhodopsin. This photobleaching is considerable even at physiological temperatures and becomes large at 50-60 degrees C. The photobleaching also increases with increasing pH from 7 to 10. We suggest that the bleaching at its final stage could be due to the dissociation of the retinal and a local thermal denaturation-like process. These facts may question the generally held belief that BR is a stable protein in vivo for a long period of time. Our results may have relevance also to practical applications of bacteriorhodopsin where the stability of bacteriorhodopsin is a key issue. In certain instances, the use of bacteriorhodopsin may require cooled conditions. Here, we defined the conditions under which bacteriorhodopsin is stable. The permanent photobleaching offers a new way of picture imaging and information input for bacteriorhodopsin-based optical devices.

Bacteriorhodopsins↗
PubMed1986

Steady-state ATP synthesis by bacteriorhodopsin and chloroplast coupling factor co-reconstituted into asolectin vesicles.

A method was developed for the co-reconstitution of bacteriorhodopsin and chloroplast coupling factor in asolectin vesicles. First, bacteriorhodopsin was reconstituted from a mixture of octyl glucoside, asolectin, and protein in the presence of ethylenediaminetetraacetic acid by passage through a Sephadex G-50 centrifuge column. Then, the purified coupling factor was reconstituted from a mixture of sodium cholate, bacteriorhodopsin vesicles, and coupling factor in the presence of Mg2+ by passage through the centrifuge column. Sucrose density-gradient centrifugation indicated a band of vesicles with slightly different positions in the gradient for maximum vesicle concentration, bacteriorhodopsin vesicle concentration, ATP synthesis, and ATP hydrolysis. The rate of light-driven ATP synthesis reaches a limiting value as the concentration of bacteriorhodopsin and the light intensity are increased. A steady-state rate of ATP synthesis of 1 mumol per mg of coupling factor X min-1 has been achieved. Apparently this rate is limited by the heterogeneity within the vesicle population and by the ability of bacteriorhodopsin to form a sufficiently large pH gradient.

Adenosine Triphosphatases↗
PubMed1990

Effect of genetic modification of tyrosine-185 on the proton pump and the blue-to-purple transition in bacteriorhodopsin.

The retinylidene chromophore mutant (Y185F) of bacteriorhodopsin, in which Tyr-185 is substituted by phenylalanine, is examined and compared with wild-type bacteriorhodopsin expressed in Escherichia coli; both were reinstituted similarly in vesicles. The Y185F mutant shows (at least) two distinct spectra at neutral pH. Upon light absorption, the blue species (which absorbs in the red) behaves as if "dead"--i.e., neither its tyrosine nor its protonated Schiff base undergoes deprotonation nor does its tryptophan fluorescence undergo quenching. This result is unlike either the purple species (which absorbs in the blue) or wild-type bacteriorhodopsin expressed in E. coli. As the pH increases, both the color changes and the protonated Schiff base deprotonation efficiency suggest a blue-to-purple transition of the Y185F mutant near pH 9. If this blue-to-purple transition of Y185F corresponds to the blue-to-purple transition of purple-membrane (native) bacteriorhodopsin (occurring at pH 2.6) and of wild-type bacteriorhodopsin expressed in E. coli (occurring at pH 5), the protein-conformation changes of this transition as well as the protonated Schiff base deprotonation may be controlled not by surface pH alone, but rather by the coupling between surface potential and the general protein internal structure around the active site. The results also suggest that Tyr-185 does not deprotonate during the photocycle in purple-membrane bacteriorhodopsin.

Bacteriorhodopsins↗
PubMed1981

Fast stages of photoelectric processes in biological membranes. I. Bacteriorhodopsin.

Bacteriorhodopsin-containing fragments of Halobacterium halobium membrane (bacteriorhodopsin sheets) were incorporated into a lecithin-impregnated collodion film, and fast stages of flash-induced electrogenesis were measured by two electrodes separated by this film. It is found that a single turnover of bacteriorhodopsin results in an electrogenic response composed of three main stages of the following tau: the first less than 200 ns, the second 15 - 70 microseconds and the third 10 ms. The second and third phases are of the same direction as an electric response to continuous illumination, whereas the first one is oppositely directed. The microseconds and ms stages were shown to correlate, in the first approximation, with formation and decomposition of the bacteriorhodopsin intermediate absorbing with 412 nm, respectively. Both the second and third phases of the photoelectric response are sums of at least two exponents. The third stage is specifically inhibition by La3+ ions which are also shown to decrease the rate of regeneration of the original bacteriorhodopsin absorbing at 570 nm from the intermediate absorbing at 412 nm. Acidification of the medium induces parallel inhibition of the second and third phases and of formation of the intermediate absorbing at 412 nm as if protonation of a group with pK = 3.6 were responsible for this inhibition. The first (opposite) phase survives acidification. It even increases at pH lower than 1.5. At such a low pH, one can show a good correlation of decays of photopotential and of a bacteriorhodopsin bathointermediate. The decays are biphasic (tau 1 = 200 microseconds and tau 2 = 2 ms), formation of both the photopotential and the bathointermediate being faster than 200 ns. At higher pH, when a three-phase photoelectric response is revealed, decay of the formed electric potential difference gives the average tau value of about 1 s. It can be accelerated by compounds that increase ionic conductance of biomembranes. At pH below 4, fluoride is found to completely inhibit the second and third phases, so that only the first phase is observed. The results are discussed in terms of a scheme postulating that the first electrogenic phase is a result of translocation of the protonated Schiff base inside the membrane due to a light-induced conformation change in retinal or protein. The second and third phases are explained by H+ transfer from the Schiff base to the outer membrane surface and from inner (cytoplasmic) surface of membrane to the Schiff base, respectively.

Bacteriorhodopsins↗
PubMed1984

Bacteriorhodopsins with chromophores modified at the beta-ionone site. Formation and light-driven action of the proton pump.

The binding to bacterioopsin of the all-trans isomers of retinal analogues lacking the six-membered ring and differing in length of the conjugated chain, as well as the light-driven action of the proton pump of the resulting bacteriorhodopsin analogues, were studied. The 'opsin shifts' in these modified bacteriorhodopsins are all around 2700 cm-1 and do not depend on the number of double bonds in the chromophore. These experimental results suggest that the 4800 cm-1 'opsin shift' in unmodified bacteriorhodopsin consists of a contribution of about 2700 cm-1 due to the interaction of the protonated Schiff-base with the counterion. The extra 2100 cm-1 shift in bacteriorhodopsin is due to the specific interaction of the cyclohexene ring and the protein. Only the bacteriorhodopsin analogue with the same number of conjugated double bonds in the chromophore as bacteriorhodopsin itself shows light-driven proton pump action.

Bacteriorhodopsins↗
PubMed1987

Energy-linked nicotinamide-nucleotide transhydrogenase. Light-driven transhydrogenase catalyzed by transhydrogenase from beef heart mitochondria reconstituted with bacteriorhodopsin.

Purified nicotinamide-nucleotide transhydrogenase from beef heart mitochondria was co-reconstituted with bacteriorhodopsin to from transhydrogenase-bacteriorhodopsin vesicles that catalyze a 20-fold light-dependent and uncoupler-sensitive stimulation of the reduction of NADP+ and NADP+ analogs by NADH and a 50-fold shift of the nicotinamide nucleotide ratio. In the presence of light, the transhydrogenase-bacteriorhodopsin vesicles catalyzed a pronounced light intensity-dependent inward proton pumping as indicated by a pH shift of the medium. As indicated by pH shifts, proton pumping by the bacteriorhodopsin essentially paralleled the light-driven transhydrogenase. Addition of valinomycin increased the pH shift twice with a concomitant 50% inhibition of the light-driven transhydrogenase, whereas nigericin inhibited the pH shift completely and the light-driven transhydrogenase partially. Taken together, these results suggest that transhydrogenase and bacteriorhodopsin interact through a delocalized proton-motive force. Possible partial reactions of transhydrogenase were investigated with transhydrogenase-bacteriorhodopsin vesicles energized by light. Reduction of oxidized 3-acetylpyridine adenine dinucleotide by NADH, previously claimed to represent partial reactions, was found to require NADPH. Similarly, reduction of thio-NADP+ by NADPH required NADH. It is concluded that these reactions do not represent partial reactions.

Adenosine Triphosphate↗
PubMed1987

Monomer-oligomer equilibrium of bacteriorhodopsin in reconstituted proteoliposomes. A freeze-fracture electron microscope study.

An improved freeze-fracture electron microscope procedure has been developed and applied to the study of the association of bacteriorhodopsin in large proteoliposomes reconstituted by reverse-phase evaporation with egg lecithin. Due to the improved accuracy and resolution of this procedure, intramembrane particles, the diameter of which (4.5 nm) closely matched that of bacteriorhodopsin monomer, could be observed at high lipid to protein ratios (greater than or equal to 40 w/w). At lower lipid to protein ratios, larger particles (diameter 7.5 nm) progressively appeared, resulting in bimodal particle size distributions up to a lipid to protein ratio of 1, where the large particles were the sole species present. These large particles were interpreted as corresponding to bacteriorhodopsin oligomers. Because of the large size and homogeneity of proteoliposomes, accurate particle density measurements could be performed. These confirmed the occurrence of a lipid to protein ratio-dependent bacteriorhodopsin monomer-oligomer equilibrium and further allowed us to identify the oligomer as a trimer or a tetramer. In complementary experiments, it was found that the bacteriorhodopsin monomer and oligomer had identical visible CD spectra and light-induced proton pumping rates. However, a large increase of the proton passive leak rate of proteoliposomes was found to be associated with oligomer formation. The appearance of these oligomers may be important as the first step in the formation of two-dimensional crystals of bacteriorhodopsin.

Bacteriorhodopsins↗
PubMed1984

Regeneration of native bacteriorhodopsin structure from fragments.

The previously described chymotryptic fragment of bacteriorhodopsin, C-2 (amino acids 1-71), is cleaved by 70% formic acid to two fragments, A-1 (amino acids 1-36) and A-2 (amino acids 37-71), which have been separated by high pressure liquid chromatography. The fragments A-1 and A-2, separately or together, are not able to replace C-2 in forming a stable bacteriorhodopsin-like complex with the fragment C-1 (amino acids 72-248) and all-trans-retinal. A second set of bacteriorhodopsin fragments, B-1 (amino acids 1-155) and B-2 (amino acids 156-248), have been prepared by sodium borohydride cleavage of bacteriorhodopsin. Following denaturation, fragments B-1 and B-2 reassociate in the presence of retinal to regenerate the native bacteriorhodopsin chromophore (approximately 40%). Fragment B-1 also interacts with fragment C-1 and all-trans-retinal to form a complex with spectral properties and secondary structure similar to those of bacteriorhodopsin. Vesicles prepared from the reconstituted fragment complexes (B-1 + B-2) or (B-1 + C-1), show proton pumping activities comparable to the previously described activity fragments C-1 and C-2.

Bacteriorhodopsins↗
PubMed1982

Denaturation and renaturation of bacteriorhodopsin in detergents and lipid-detergent mixtures.

The denatured and renatured states of bacteriorhodopsin have been studied in detergents and lipid/detergent mixtures by using ultraviolet and visible light absorption spectroscopy, fluorescence spectroscopy, and circular dichroism. Upon solubilization in sodium dodecyl sulfate, bacteriorhodopsin undergoes denaturation with partial loss of the secondary structure and loss of retinal binding ability. In contrast, delipidated bacteriorhodopsin retains its native structure in deoxycholate and undergoes denaturation only on photobleaching, which results in a partial loss of the secondary structure. Native secondary structure spontaneously reforms upon addition of phospholipid/cholate mixtures to bacterio-opsin denatured in sodium dodecyl sulfate. Upon subsequent addition of retinal, up to 95% of the native chromophore (lambdamax = 550-560 nm) is restored. Renaturation also restores the characteristics of light and dark adaptation and tight retinal binding (Kb greater than 10(7) M-1). Renaturation of delipidated bacteriorhodopsin denatured in sodium dodecyl sulfate also occurs without phospholipid upon the addition of retinal and Triton X-100 or octyl glucoside. The equilibrium between monomeric and oligomeric states of bacteriorhodopsin has been studied by circular dichroism and fluorescence quenching. Bacteriorhodopsin renatured with Triton X-100 or phospholipid/cholate is monomeric. However, removal of the detergents from renatured protein under specific conditions results in formation of an oligomer in which quaternary interactions are regenerated. The application of fluorescence quenching to the determination of oligomeric size in intrinsic membrane proteins is discussed.

Bacteriorhodopsins↗
PubMed1993

Orthorhombic crystal form of bacteriorhodopsin nucleated on benzamidine diffracting to 3.6 A resolution.

Freshly formed benzamidine crystals were found to provide a suitable nucleation surface for crystallisation of bacteriorhodopsin. At 20 degrees C and 1% octylglucoside pseudohexagonal needles of bacteriorhodopsin nucleated on the benzamidine surface. At 4 degrees C and reduced detergent concentration (0.5%) a new, better-ordered orthorhombic crystal form of bacteriorhodopsin was formed by heterogeneous nucleation on benzamidine. Polarised absorption spectroscopy and flash photolysis experiments were used to show that the crystalline bacteriorhodopsin is photoactive in both forms. The M-intermediate absorbs maximally at 405nm and formation of M does not disturb the crystal lattice. The plate-shaped crystals diffract to a resolution of 3.6 A along the a and b directions and to 4.2 A in the c direction. The most likely space group of the crystals is C222 (a = 107.5 A, b = 117.0 A, c = 69.5 A). The crystals are built from layers of bacteriorhodopsin molecules that are tilted from the c axis by about 45 degrees. In addition, as a background to the discovery of the new crystal form, the influence of different detergents, additives and precipitants on the formation of pseudohexagonal needles is presented.

Bacteriorhodopsins↗
PubMed2000

The final stages of folding of the membrane protein bacteriorhodopsin occur by kinetically indistinguishable parallel folding paths that are mediated by pH.

The folding of the transmembrane protein bacteriorhodopsin that occurs during the binding of its retinal cofactor is investigated in a membrane-like environment. Changes in the retinal absorption band reveal two transient retinal-protein intermediate states, with apparent absorption maxima at 380 nm and 440 nm, respectively. Studies on a bacteriorhodopsin mutant of Lys216, which cannot bind retinal covalently, add to evidence that retinal is non-covalently bound in these intermediate states. The two retinal-protein intermediates are genuine intermediate states that form in parallel, each with an observed rate constant of 1.1 s-1. Meanwhile no formation of the folded state is detected. Folded bacteriorhodopsin, with all trans retinal covalently bound, forms from both retinal-bound intermediates with the same apparent rate constant of 0.0070 s-1 that is independent of retinal concentration. Retinal isomerisation then occurs with a rate constant of 0.00033 s-1 to give bacteriorhodopsin containing all trans and 13 cis-retinal. These results provide experimental evidence for multiple folding routes for a membrane protein that are pH dependent, with pH conditions determining the apparent folding route. These observed parallel folding paths are kinetically indistinguishable, which contrasts with most other observations of parallel folding pathways where only pathways with different kinetics have been reported. Furthermore, together with previous work, this study shows that bacteriorhodopsin has to populate at least two folding intermediates, during folding in the mixed lipid micelles investigated here, before the final fold is attained.

Bacteriorhodopsins↗
PubMed1990

Peptide building blocks from bacteriorhodopsin: isolation and physicochemical characterization of two individual transmembrane segments.

For protein engineering purposes, transmembrane segments of the structurally stable protein bacteriorhodopsin have been isolated and chemically characterized. Bacteriorhodopsin was cleaved by protease V8 from Staphylococcus aureus to two fragments, V-1 and V-2. The V-2 fragment was separated by gel filtration in organic solvents and purified by reversed-phase FPLC. The fragment has been identified as the C-terminal, partially truncated double-loop of bacteriorhodopsin, including amino acids Val-167-Glu-232/4. Cleavage of V-2 by cyanogen bromide at the single Met-209 yielded two subfragments, which were purified to homogeneity by FPLC procedures. The N-terminal subfragment psi, consisted of a single transmembrane segment (helix F) of bacteriorhodopsin (Val-167-Met(Hse)-209). The C-terminal amphipathic subfragment omega, (Val-210-Glu-232/4) was identified as part of the C-terminal seventh helix of bacteriorhodopsin. Secondary structures of V-2, psi, and omega were investigated in organic solvents and micellar solutions. Native helical structures were partially retained in the solvent systems mentioned.

Amino Acid Sequence↗
PubMed1977

Proton transport by bacteriorhodopsin through an interface film.

Interface films of purple membrane and lipid containing spectroscopically intact and oriented bacteriorhodopsin have been used as a model system to study the function of this protein. Small positive charges in surface potential (less than 1 mV) are detected upon illumination of these films at the air-water interface. These photopotentials are not affected by overlaying the interface film with a thin layer (0.3 mm) of decane. However, they are dramatically increased when lipid soluble proton carriers FCCP or DNP are added to the decane. The polarity of the photopotential indicates that, in the light, positive charges are transported through the interface from the aqueous to the organic phase. The action spectrum of the photopotential is identical to the absorption spectrum of bacteriorhodopsin. Since bacteriorhodopsin molecules are oriented with their intracellular surface towards the aqueous subphase, the characteristics of the photopotential indicate that in the light bacteriorhodopsin translocates protons from its intracellular to its extracellular surface. The kinetics of the photopotential reveal that the rate and extent of proton transport are proportional both to the fraction of bacteriorhodopsin molecules excited and to the concentration of proton acceptor. The photopotentials result from changes in the ionic distribution across the decane-water interface and can be cancelled by lipid soluble anions.

Air↗
PubMed2001

The role of retinal in the long-range protein-lipid interactions in bacteriorhodopsin-phosphatidylcholine vesicles.

The effects of bacteriorhodopsin analogues and the analogues of a bacteriorhodopsin mutant (D96N) on the lateral organization of lipids have been investigated with lipid species with a variety of acyl chain lengths. The analogues, obtained by regeneration of bacterioopsin or mutant opsin with 14-, 12-, 10-, or 8-fluororetinal, were reconstituted with 1,2-didodecanoyl-sn-glycero-3-phosphocholine, 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine, 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine, and 1,2-dioctadecanoyl-sn-glycero-3-phosphocholine. The phase behavior of the protein-lipid systems was investigated at different temperatures and different protein/lipid molar ratios by analyzing the fluorescence and phase properties of the 1-acyl-2-[8-(2-anthroyl)octanol]-sn-glycero-3-phosphocholine probe. The (8,10,12)-bacteriorhodopsins had a similar effect on the lipid phase transition to that induced by native bacteriorhodopsin: a rigidifying effect on the three shorter lipid species and a fluidifying effect on the longest-chain lipids used. The substitution of retinal with 14-fluororetinal resulted in much stronger effects of the protein on the lipids: a more pronounced up-shift of the lipid phase transition temperature, a rigidifying effect on all the lipids used, and an elongation of the distance over which the hydrophobic thickness of the lipid bilayer was perturbed by the protein. Evidence was provided that retinal contributed to the long-range protein-lipid interactions in bacteriorhodopsin-phosphatidylcholine vesicles. The extent of this contribution was dependent on the retinal structure in close vicinity to the Shiff base and on the compactness of the protein structure.

Bacteriorhodopsins↗
PubMed1978

Mechanism of generation and regulation of photopotential by bacteriorhodopsin in bimolecular lipid membrane.

Photoelectric properties of bacteriorhodopsin incorporated into a bimolecular lipid membrane were investigated with special regard to the mechanism of photoelectric field generation. It was shown that besides its proton pump and electric generator functions bacteriorhodopsin works as a possible molecular regulator of the light-induced membrane potential. When a bimolecular lipid membrane containing bacteriorhodopsin is continuously illuminated in its main visible absorption band, and afterwards by superimposed blue light matching the absorption band of the long-living photobleached bacteriorhodopsin (M412) as well, the latter either enhances or decreases the steady-state photoresponse, depending upon the intensity of the green light. Thus, the additional blue-light illumination tends to cause the resultant photoelectric membrane potential to become stabilized. Two alternative schemes are tentatively proposed for the photochemical cycle of bacteriorhodopsin whereby blue light can control photovoltage generation. A kinetic model of the proton pump and the regulation of the photoelectric membrane potential is presented. This model fits all the experimental findings, even quantitatively. From the model some kinetic and physical parameters of this light-driven pump could be determined.

Bacteriorhodopsins↗
PubMed1978

Transient photovoltages in purple membrane multilayers. Charge displacement in bacteriorhodopsin and its photointermediates.

The photovoltaic properties of bacteriorhodopsin molecules and their photochemical intermediates have been investigated in an experimental cell consisting of multilayered films of highly oriented, dry fragments of purple membrane and lipid sandwiched between two metal (Pd) electrodes. The electrical time constant of these sandwich cells containing between 5 and 30 layers is less than 10(-5) S. Bright illumination of these cells with actinic flashes of approximately 1 ms duration generates transient photovoltages. These photovoltages, which make the extracellular surface of purple membrane positive with respect to the intracellular surface, follow the time course of the flash with no detectable latency. The amplitude of the photovoltages increases linearly with light intensity and their action spectrum matches the absorption spectrum of the light-adapted state of bacteriorhodopsin, BR570. In these dry multilayer cells, the slow photointermediates of bacteriorhodopsin, M412, N520 and O640 are long lived. Illumination of the sandwich cells with long duration (200 ms) pulses of light results, therefore, in the formation of photomixtures containing all these slow photointermediates. Flash illumination of the sandwich cells immediately following the conditioning pulse produces photovoltages whose action spectra match the absorption spectra of the M412 and N520 photointermediates. The M412 photovoltages, like the BR570 photovoltages, follow the time course of the actinic flash with no detectable latency and increase in amplitude linearly with light intensity. But, unlike the BR570 photovoltage, the M412, N520 and O640 photovoltages make the extracellular surface of purple membrane negative with respect to the intracellular surface. Through the of their specific photovoltaic signals, M412 and N520 are shown to be kinetically distinct photointermediates of bacteriorhodopsin. Detection of fast photovoltages with these characteristics in the absence of any ionic solution, and in parallel with spectrophotometric changes, suggest that they arise from charge displacements in the bacteriorhodopsin molecules and their photointermediates as they undergo photochemical conversion in response to the absorption of photons.

Bacteriorhodopsins↗
PubMed1994

On the two forms of bacteriorhodopsin.

In our previous work [(1993) FEBS Lett. 313, 248-250; (1993) Biochem. Int. 30, 461-469] M-intermediate formation of wild-type bacteriorhodopsin was shown to involve two components differing in time constants (tau 1 = 60-70 microseconds and tau 2 = 220-250 microseconds), which were suggested to reflect two independent pathways of M-intermediate formation. The contribution of the fast M was 4-times higher than the slow one. Our present research on M-intermediate formation in the D115N bacteriorhodopsin mutant revealed the same components but at a contribution ratio of 1:1. Upon lowering the pH, the slow phase of M-formation vanished at a pK of 6.2, and in the pH region 3.0-5.5 only the M-intermediate with a rise time of 60 microseconds was present. A 5-6 h incubation of D115N bacteriorhodopsin at pH 10.6 resulted in the irreversible transformation of 50% of the protein into a form with a difference absorbance maximum at 460 nm. This form was stable at pH 7.5 and had no photocycle, including M-intermediate formation. The remaining bacteriorhodopsin contained 100% fast M-intermediate. The disappearance of the 250-microseconds phase concomitant with bR460 formation indicates that at neutral pH bacteriorhodopsin exists as two spectroscopically indistinguishable forms.

Bacteriorhodopsins↗