Localization of two chymotryptic fragments in the structure of renatured bacteriorhodopsin by neutron diffraction.

The structure of crystalline purple membrane reconstituted from purified bacteriorhodopsin (BR) chymotryptic fragments has been studied by neutron diffraction. In one of the samples studied, the fragment C-2, encompassing the first two predicted transmembrane segments, was prepared from deuterated purple membrane. The diffraction changes when the natural C-2 fragment is substituted by a deuterated one are analysed in terms of a seven-helix model for BR. The assignment of the labelled fragment to one end of the molecule placed new constraints on folding models for the protein.     

Refolding of bacteriorhodopsin in lipid bilayers. A thermodynamically controlled two-stage process

Possible steps in the folding of bacteriorhodopsin are revealed by studying the refolding and interaction of two fragments of the molecule reconstituted in lipid vesicles. (1) Two denatured bacteriorhodopsin fragments have been purified starting from chymotryptically cleaved bacteriorhodopsin. Cleaved bacteriorhodopsin has been renatured from a mixture of the fragments in Halobacterium lipids/retinal/dodecyl sulfate solution following removal of dodecyl sulfate by precipitation with potassium. The renatured molecules have the same absorption spectrum and extinction coefficient as native cleaved bacteriorhodopsin. They are integrated into small lipid vesicles as a mixture of monomers and aggregates. Extended lattices form during the partial dehydration process used to orient samples for X-ray and neutron crystallography. (2) Correct refolding of cleaved bacterioopsin occurs upon renaturation in the absence of retinal. Regeneration of the chromophore and reformation of the purple membrane lattice are observed following subsequent addition of all-trans retinal. (3) The two chymotryptic fragments have been reinserted separately into lipid vesicles and refolded in the absence of retinal. Circular dichroism spectra of the polypeptide backbone transitions indicate that they have regained a highly alpha-helical structure. The kinetics of chromophore regeneration following reassociation have been studied by absorption spectroscopy. Upon vesicle fusion, the refolded fragments first reassociate, then bind retinal and finally regenerate cleaved bacteriorhodopsin. The complex formed in the absence of retinal is kinetically indistinguishable from cleaved bacterioopsin. The refolded fragments in lipid vesicles are stable for months, both as separate entities and after reassociation. These observations provide further evidence that the native folded structure of bacteriorhodopsin lies at a free energy minimum. They are interpreted in terms of a two-stage folding mechanism for membrane proteins in which stable transmembrane helices are first formed. They subsequently pack without major rearrangement to produce the tertiary structure.     

NMR structural analysis of a membrane protein: bacteriorhodopsin peptide backbone orientation and motion

In reconstituted vesicles above the lipid phase transition temperature, bacteriorhodopsin (BR) undergoes rotational diffusion about an axis perpendicular to the plane of the bilayer [Cherry, R. J., Muller, U., & Schneider, G. (1977) FEBS Lett. 80, 465]. This diffusion narrows the 13C NMR powder line shape of the BR peptide carbonyls. In contrast, BR in native purple membrane is relatively immobile and exhibits a rigid-lattice powder line shape. By use of the principal values of the rigid-lattice chemical shift tensor and the motionally narrowed line shape from the reconstituted system, the range of Euler angles of the leucine peptide groups relative to the diffusion axis has been calculated. The experimentally observed line shape is inconsistent with those expected for structures which consist entirely of either alpha helix or beta sheet perpendicular to the membrane or beta sheet tilted at angles up to about 60 degrees from the membrane normal. However, for two more complex structural models, the predicted line shapes agree well with the experimental one. These are, first, a structure consisting entirely of alpha1 helices tilted at 20 degrees from the membrane normal and, second, a combination of 60% alpha II helix perpendicular to the membrane plane and 40% antiparallel beta sheet tilted at 10-20 degrees from the membrane normal. The results also indicate that the peptide backbone of bacteriorhodopsin in native purple membrane is extremely rigid even at 40 degrees. The experiments presented here demonstrate a new approach, using solid-state nuclear magnetic resonance (NMR) methods, for structural studies of transmembrane proteins in fluid membrane environments, either natural or reconstituted.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamic measurements of the contributions of helix-connecting loops and of retinal to the stability of bacteriorhodopsin.

Thermodynamic studies of bacteriorhodopsin (BR) have been undertaken in order to investigate the factors that stabilize the structure of a membrane protein. The stability of the native, intact protein was compared to that of protein with retinal removed, and/or cleaved in one or two of the loops connecting the transmembrane helices. The stability was assessed using differential scanning calorimetry and thermal denaturation curves obtained from ultraviolet circular dichroism and absorption spectroscopy. Retinal binding and the loop connections were each found to make a small contribution to stability, and even a sample that was cleaved twice as well as bleached to remove retinal denatured well above room temperature. Removal of retinal destabilized the protein more than cleaving once, and about as much as cleaving twice. Retinal binding and the connections in the loops were found to stabilize BR in independent ways. Cleavage of the molecule into fragments did not reduce the intermolecular cooperativity of the denaturation. Dilution of the protein by addition of excess lipid in order to eliminate the purple membrane crystal lattice also did not alter the cooperativity. These results are used to compare the relative importance of various contributors to the stability of BR.     

Tertiary structure of bacteriorhodopsin. Positions and orientations of helices A and B in the structural map determined by neutron diffraction

Positions and rotations of two helices in the tertiary structure of bacteriorhodopsin have been studied by neutron diffraction using reconstituted, hybrid purple membrane samples. Purple membrane was biosynthetically 2H-labeled at non-exchangeable hydrogen positions of leucine and tryptophan residues. Two chymotryptic fragments were purified, encompassing either the first two or the last five of the seven putative transmembrane segments identified in the amino acid sequence of bacteriorhodopsin. The 2H-labeled fragments, diluted to variable extents with the identical, unlabeled fragment, were mixed with their unlabeled counterpart; bacteriorhodopsin was then renatured and reconstituted. The crystalline purple membrane samples thus obtained contained hybrid bacteriorhodopsin molecules in which certain transmembrane segments had been selectively 2H-labeled to various degrees. Neutron diffraction powder patterns were recorded and analyzed both by calculating difference Fourier maps and by model building. The two analyses yielded consistent results. The first and second transmembrane segments in the sequence correspond to helices 1 and 7 of the three-dimensional structure, respectively. Rotational orientations of these two helices were identified using best fits to the observed diffraction intensities. The data also put restrictions on the position of the third transmembrane segment. These observations are discussed in the context of folding models for bacteriorhodopsin, the environment of the retinal Schiff base, and site-directed mutagenesis experiments.     

Reconstitution of bacteriorhodopsin into cyclic lipid vesicles

Bacteriorhodopsin (bR), a membrane protein that can generate a light-driven proton pump, was successfully reconstituted into vesicles composed of an artificial cyclic lipid that mimics archaeal membrane lipids. Unlike reconstituted bR in 1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles, the net topology and structure of bR molecules in cyclic lipid vesicles are identical to those in the native purple membrane of Halobacterium salinarum.     

Role of helix-helix interactions in assembly of the bacteriorhodopsin lattice.

The purple membrane of Halobacterium salinarium is a two-dimensional lattice of lipids and the integral membrane protein bacteriorhodopsin (BR). To determine whether helix-helix interactions within the membrane core stabilize this complex, we substituted amino acid residues at the helix-helix interface between BR monomers and examined the assembly of the protein into the lattice. Lattice assembly was demonstrated to fit a cooperative self-assembly model that exhibits a critical concentration in vivo. Using this model as the basis for a quantitative assay of lattice stability, bulky substitutions at the helix-helix interface between BR monomers within the membrane core were shown to be destabilizing, probably due to steric clash. Ala substitutions of two residues at the helix-helix interface also reduced stability, suggesting that the side chains of these residues participate in favorable van der Waals packing interactions. However, the stabilizing interactions were restricted to a small region of the interface, and most of the substitutions had little effect. Thus, the contribution of helix-helix interactions to lattice stability appears limited, and favorable interactions between other regions of neighboring BR monomers or between BR and lipid molecules must also contribute.     

Purification of bacteriorhodopsin and characterization of mature and partially processed forms

Bacteriorhodopsin (BR) essentially free of native lipids has been prepared in a highly stable state. Purple membrane was solubilized in Triton X-100 and BR was purified by size exclusion chromatography using 3-[cholamidopropyl)dimethylammonio]-2-hydroxyl-1-propanesulfonic acid (CHAPSO) detergent at pH 5. Molar ratios of phospholipid/BR ranged from 0.4 to 0.05 corresponding to 94-98% phospholipid removal. Purified BR has an absorbance ratio (A280nm/A548nm) of 1.5-1.6 in the dark-adapted state which is the highest purified BR/protein ratio reported to date. The purified BR in CHAPSO shows maximum stability in the pH range 5.0-5.5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of native purple membrane and solubilized BR from most Halobacterium halobium JW-3 cultures show 3 higher molecular weight bands in addition to BR. Immunological staining and amino acid sequencing indicates that these additional proteins are partially processed forms of the BR precursor protein. The BR preprotein contains 13 additional amino acids on the NH2 terminus which are removed by post-translational processing in at least four steps. Isoelectric focusing separated most delipidated and non-delipidated BR samples into 8 bands. Incomplete BR post-translational processing BR is thought to be largely responsible for the multiplicity of isoelectric BR species. The principal components have pI values of 5.20 and 5.24 and both have absorption maxima at 550 nm, characteristic of detergent-solubilized BR. BR in Triton X-100 or nonylglucoside, delipidated BR in CHAPSO, and BR in intact purple membrane all have a dark-adapted ratio of 13-cis to all-trans-retinal of 1.9:1.     

The biocatalytic effect of Halobacterium halobium on photoelectrochemical hydrogen production

Hydrogen gas can be produced electrochemically by leading a current through two electrodes immersed in a NaCl solution. Bacteriorhodopsin (BR) a protein found in the purple membrane of Halobacterium halobium, is known to pump protons across the membrane upon illumination. In this study, the effect of BR on photoelectrochemical hydrogen production was investigated. A batch type bio-photoelectrochemical reactor was designed and constructed. The photoelectrochemical hydrogen production experiments were performed with free H. halobium packed cells or immobilised H. halobium cells. The cells were either immobilised in polyacrylamide gel (PAG) or on cellulose acetate membrane (CAM). Experiments were also performed with purple membrane fragments of H. halobium immobilised on cellulose acetate membrane. It was found that the presence of bacteriorhodopsin (BR) in the reactor enhances the hydrogen production rate upon illumination. Immobilisation increased the amount of hydrogen produced per mole of BR. Compared to control experiments without BR, the power requirement of the photoelectrochemical reactor per amount of hydrogen produced decreased fourfold when purple membrane fragments immobilised on CAM were used. The presence of BR regulates the pH of the system, increases the hydrogen production rate and causes light-induced proton dissociation, which lowers the electrical power requirement for the electrochemical conversion.     

Stability of the two-dimensional lattice of bacteriorhodopsin reconstituted in partially fluorinated phosphatidylcholine bilayers

This study aims to investigate bacteriorhodopsin (bR) molecules reconstituted in lipid bilayers composed of di(nonafluorotetradecanoyl)-phosphatidylcholine (F4-DMPC), a partially fluorinated analogue of dimyristoyl-phosphatidylcholine (DMPC) to clarify the effects of partially fluorinated hydrophobic chains of lipids on protein's stability. Calorimetry measurements showed that the chain-melting transition of F4-DMPC/bR systems occurs at 3.5 °C, whereas visible circular dichroism (CD) and X-ray diffraction measurements showed that a two-dimensional (2D) hexagonal lattice formed by bR trimers in F4-DMPC bilayers remains intact even above 30 °C, similar to bR in a native purple membrane. Complete dissociation of the trimers into the monomers detected by visible CD almost coincides with the complete melting of 2D lattice observed by X-ray diffraction, in which both take place at around 65 °C (10 °C lower than that for bR in a native purple membrane). However, it is extremely high in comparison with the bR reconstituted in DMPC bilayers in which the dissociation of bR trimer in DMPC bilayers occurs near the chain-melting transition temperature of DMPC bilayers at approximately 18 °C. In order to explore the rationale behind the difference in stability, a further investigation of the detailed structural features of pure F4-DMPC bilayers was performed by analyzing the lamellar diffraction data using simple electron density models. The results suggested that the perfluoroalkyl groups do not exhibit any conformation change even if the chain-melting transition occurs, which is likely to contribute to the stability of the 2D hexagonal lattice formed by the bR trimers.     

A Monte Carlo study of the self-assembly of bacteriorhodopsin

Bacteriorhodopsin (BR) and specific lipid molecules self-assemble into a quasi two-dimensional lattice structure known as the purple membrane (PM). In the PM, BR molecules exist in a trimeric form with lipid molecules present in the space enclosed by each trimeric unit and in the inter-trimer space. These trimeric units, which have a roughly circular cross-section, are arranged in hexagonal patterns with long-ranged crystalline order. In this work, we investigate the self-assembly of BR in the PM via Monte Carlo simulations of a two-dimensional model of the membrane and proteins. The protein molecules are modeled as 120 degrees sectors of a circle and the lipid molecules enter into the model through effective protein-protein interactions. The sectors cannot overlap with each other, and in addition to this excluded volume interaction there are site-site attractive interactions between specific points of the proteins to mimic interactions between helices on the proteins and lipid-induced interactions. At low values of the attractive well depth, the proteins are found in the monomeric form at all concentrations. At moderate and high values of the attractive well depth, trimers are formed as the concentration increases, and with a further increase in concentration the trimers organize into a hexagonal lattice. The interactions between the proteins and those induced by the intra-trimer lipids play an equally important role in the formation of trimers and the lattice. The lipids in the inter-trimer space cause the trimers to orient in a specific direction in the hexagonal crystal lattice.     

Bacteriorhodopsin can be refolded from two independently stable transmembrane helices and the complementary five-helix fragment.

This paper describes experimental tests of the hypothesis that bacteriorhodopsin (BR) can fold by the association of independently stable transmembrane helices. Peptides containing the first and second helical segments of BR were chemically synthesized. These two peptides and the complementary five-helix fragment of BR were reconstituted in three separate populations of native-lipid vesicles which were then mixed and fused to allow the fragments to interact. After addition of retinal, absorption spectroscopy of the reconstituted BR and X-ray diffraction of two-dimensional crystals of this material showed that the native structure of BR was regenerated. The first two helices of BR can therefore be considered as independent folding domains, and covalent connections in the loops connecting the helices to each other and to the rest of the molecule are not essential for the appropriate association of the helices.     

The chromophore retinal hinders passive proton/hydroxide ion translocation through bacteriorhodopsin

Experiments have been performed to examine any influence of the chromophore retinal in bacteriorhodopsin (BR) on the passive proton/hydroxide ion flux through this integral membrane protein. BR was reconstituted into dimyristoylphosphatidylcholine (DMPC)-phosphatidylserine or DMPC-dimyristoylphosphatidylglycerol unilamellar vesicles with molar lipid to protein ratios ranging from 30 to 150. The entrapped fluorescence dye pyranine served as a reliable indicator of the internal proton concentration. Transmembrane pH-gradients were quickly established across the vesicular membrane and the kinetics of the induced fluorescence changes were compared for vesicles with incorporated native BR, BR bleached to the chromophore-free protein bacterioopsin, and BR regenerated from bacterioopsin with all-trans-retinal, respectively. For aggregated protein molecules, the H+/OH- diffusion across bacterioopsin was always considerably faster than that through the protein containing covalently bound retinal. The decay rate of the imposed pH-gradient was 4.4-9.1 and 2.0-5.1 times slower for native and regenerated BR, respectively, as compared to bacterioopsin. Stepwise regeneration of bacterioopsin with all-trans-retinal revealed a linear dependence of the predominant delta pH-decay time on the degree of regeneration. Essentially the same observations were made with monomeric protein molecules in vesicular lipid membranes. The results demonstrate that the chromophore retinal itself blocks the H+/OH- conducting pathway across the transmembrane protein BR or indirectly controls this path by inducing conformational changes in the protein upon binding.     

Imaging the membrane protein bacteriorhodopsin with the atomic force microscope.

The membrane protein bacteriorhodopsin was imaged in buffer solution at room temperature with the atomic force microscope. Three different substrates were used: mica, silanized glass and lipid bilayers. Single bacteriorhodopsin molecules could be imaged in purple membranes adsorbed to mica. A depression was observed between the bacteriorhodopsin molecules. The two dimensional Fourier transform showed the hexagonal lattice with a lattice constant of 6.21 +/- 0.20 nm which is in agreement with results of electron diffraction experiments. Spots at a resolution of approximately 1.1 nm could be resolved. A protein, cationic ferritin, could be imaged bound to the purple membranes on glass which was silanized with aminopropyltriethoxysilane. This opens the possibility of studying receptor/ligand binding under native conditions. In addition, purple membranes bound to a lipid bilayer were imaged. These images may help in interpreting results of functional studies done with purple membranes adsorbed to black lipid membranes.     

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.     

Lipid-Induced Modulation of the Protein Packing in Two-Dimensional Crystals of Bacteriorhodopsin

The mechanism by which bacteriorhodopsin (BR), the light-driven proton pump from the purple membrane (PM) of Halobacterium halobium, arranges in a 2D hexagonal array has been studied by reconstitution of BR in complexes of two types of bilayer made either with PM-derived lipids or with PM lipids and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). The unit cell dimensions of the 2D protein crystals, determined by correlation averaging analysis of freeze-fracture electron micrographs, were compared with the lattice constant of the PM. In complexes made with delipidated BR and with the polar lipids extracted from H. halobium cells (HHPL), BR trimers are arranged in a hexagonal lattice with the same lattice constant of 5.9 ± 0.2 nm as found in the PM. In BR-containing complexes made with PM-derived lipids and DMPC at several protein:lipid mole ratios, BR trimers are also arranged in a hexagonal lattice, but with a unit cell dimension of 9.2 ± 0.2 nm, which is about one-third larger compared to that measured in PM (Michel et al. , 1980). In a subclass of this type of complexes, orthogonal BR arrays were observed with a lattice constant of 5.9 × 9.9 ± 0.2 nm. It appears that insertion of DMPC into the BP/PM-derived lipid complexes increases the center-to-center distances in both array types by a discrete amount.     

Bacteriorhodopsin/amphipol complexes: structural and functional properties

The membrane protein bacteriorhodopsin (BR) can be kept soluble in its native state for months in the absence of detergent by amphipol (APol) A8-35, an amphiphilic polymer. After an actinic flash, A8-35-complexed BR undergoes a complete photocycle, with kinetics intermediate between that in detergent solution and that in its native membrane. BR/APol complexes form well defined, globular particles comprising a monomer of BR, a complete set of purple membrane lipids, and, in a peripheral distribution, ∼2 g APol/g BR, arranged in a compact layer. In the absence of free APol, BR/APol particles can autoassociate into small or large ordered fibrils.     

The essential role of specific Halobacterium halobium polar lipids in 2D-array formation of bacteriorhodopsin.

The mechanism whereby bacteriorhodopsin (BR), the light driven proton pump from the purple membrane of Halobacterium halobium, arranges in a 2D-hexagonal array, has been studied in bilayers containing the protein, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and various fractions of H. halobium membrane lipids, by freeze fracture electron microscopy and examination of optical diffractograms of the micrographs obtained. Electron micrographs of BR/DMPC complexes containing the entire polar lipid component of H. halobium cell membranes or the total lipid component of the purple membrane, with a protein-to-total lipid molar ratio of less than 1:50 and to which 4 M NaCl had been added, revealed that trimers of BR formed into an hexagonal 2D-array similar to that found in the native purple membrane, suggesting that one or more types of the purple membrane polar lipids are required for array formation. To support this suggestion, bacteriorhodopsin was purified free of endogenous purple membrane lipids and reconstituted into lipid bilayer complexes by detergent dialysis. The lipids used to form these complexes are 1,2-dimyristoyl-sn-glycerol-phosphocholine (DMPC) as the major lipid and, separately, each of the individual lipid types from the H. halobium cell membranes, namely 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol 1'-phosphate (DPhPGP), 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol 1'-sulphate (DPhPGS), 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol (DPhPG) and 2,3-di-O-phytanyl-1-O-[beta-D-Galp-3-sulphate-(1----6)-alpha-D- Manp-(1----2)-alpha-D-Glcp]-sn-glycerol (DPhGLS). When examined by freeze-fracture electron microscopy, only the complexes containing 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol- 1'-phosphate or 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol-1'-sulphate, at high protein density (less than 1:50, bacteriorhodopsin/phospholipid, molar ratio) and to which 4 M NaCl had been added, showed well defined 2D hexagonal arrays of bacteriorhodopsin trimers similar to those observed in the purple membrane of H. halobium.     

Influence of membrane lipids on the photochemistry of bacteriorhodopsin in the purple membrane of Halobacterium halobium

Purple membrane fragments from Halobacterium halobium were reconstituted with the native lipids replaced by dipalmitoyl phosphatidylcholine and by egg lecithin. In parallel studies the temperature dependence of bacteriorhodopsin phototransient lifetime and absorption dichroism and of in situ lipid microviscosity were determined; the former two by, respectively, conventional and polarization flash photometry, and the latter by observation of emission depolarization of an embedded fluorescent dye, 1,6-diphenyl-1,3,5-hexatriene. Discontinuities in lipid microviscosity profiles in native and egg lecithin purple membrane were reflected in both the photochemical cycle frequency and bacteriorhodopsin chromophore rotational mobility. The influence exerted by membrane-lipid viscosity appears to be a secondary effect, and points to the bacteriorhodopsin chromophoric group being situated in the protein interior.     

Purple-to-blue transition of bacteriorhodopsin in a neutral lipid environment.

The red shift in the absorption maximum of native purple membrane suspensions caused by deionization is missing in lipid-depleted purple membrane, and the pK of the acid-induced transition is down-shifted to pH approximately 1.4 and has become independent of cation concentration (Szundi, I., and W. Stoeckenius. 1987. Proc. Natl. Acad. Sci. USA. 84:3681-3684). However, the proton pumping function cannot be demonstrated in these membranes. When native acidic lipids of purple membrane are exchanged for egg phosphatidylcholine or digalactosyldiglyceride, bacteriorhodopsin is functionally active in the modified membrane. It shows spectral shifts upon light-dark adaptation, a photocycle with M-intermediate and complex decay kinetics; when reconstituted into vesicles with the same neutral lipids, it pumps protons. Unlike native purple membrane, lipid-substituted modified membranes do not show a shift of the absorption maximum to longer wavelength upon deionization. A partial shift can be induced by titration with HCl; it has a pK near 1.5 and no significant salt dependence. Titration with HNO3 and H2SO4, which causes a complete transition in the lipid-depleted membranes, i.e., it changes their colors from purple to blue, does not cause the complete transition in the lipid-substituted preparations. These results show that the purple color of bacteriorhodopsin is independent of cations and their role in the purple-to-blue transition of native membranes is indirect. The purple and blue colors of bacteriorhodopsin are interpreted as two conformational states of the protein, rather than different protonation states of a counterion to the protonated Schiff base.