ESR - a retinal protein with unusual properties from Exiguobacterium sibiricum.

This review covers the properties of a retinal protein (ESR) from the psychrotrophic bacterium Exiguobacterium sibiricum that functions as a light-driven proton pump. The presence of a lysine residue at the position corresponding to intramolecular proton donor for the Schiff base represents a unique structural feature of ESR. We have shown that Lys96 successfully facilitates delivery of protons from the cytoplasmic surface to the Schiff base, thus acting as a proton donor in ESR. Since proton uptake during the photocycle precedes Schiff base reprotonation, we conclude that this residue is initially in the uncharged state and acquires a proton for a short time after Schiff base deprotonation and M intermediate formation. Involvement of Lys as a proton donor distinguishes ESR from the related retinal proteins - bacteriorhodopsin (BR), proteorhodopsin (PR), and xanthorhodopsin (XR), in which the donor function is performed by residues with a carboxyl side chain. Like other eubacterial proton pumps (PR and XR), ESR contains a histidine residue interacting with the proton acceptor Asp85. In contrast to PR, this interaction leads to shift of the acceptor's pKa to more acidic pH, thus providing its ability to function over a wide pH range. The presence of a strong H-bond between Asp85 and His57, the structure of the proton-conducting pathways from cytoplasmic surface to the Schiff base and to extracellular surface, and other properties of ESR were demonstrated by solving its three-dimensional structure, which revealed several differences from known structures of BR and XR. The structure of ESR, its photocycle, and proton transfer reactions are discussed in comparison with homologous retinal proteins.

Aspartate-histidine interaction in the retinal schiff base counterion of the light-driven proton pump of Exiguobacterium sibiricum.

One of the distinctive features of eubacterial retinal-based proton pumps, proteorhodopsins, xanthorhodopsin, and others, is hydrogen bonding of the key aspartate residue, the counterion to the retinal Schiff base, to a histidine. We describe properties of the recently found eubacterium proton pump from Exiguobacterium sibiricum (named ESR) expressed in Escherichia coli, especially features that depend on Asp-His interaction, the protonation state of the key aspartate, Asp85, and its ability to accept a proton from the Schiff base during the photocycle. Proton pumping by liposomes and E. coli cells containing ESR occurs in a broad pH range above pH 4.5. Large light-induced pH changes indicate that ESR is a potent proton pump. Replacement of His57 with methionine or asparagine strongly affects the pH-dependent properties of ESR. In the H57M mutant, a dramatic decrease in the quantum yield of chromophore fluorescence emission and a 45 nm blue shift of the absorption maximum with an increase in the pH from 5 to 8 indicate deprotonation of the counterion with a pK(a) of 6.3, which is also the pK(a) at which the M intermediate is observed in the photocycle of the protein solubilized in detergent [dodecyl maltoside (DDM)]. This is in contrast with the case for the wild-type protein, for which the same experiments show that the major fraction of Asp85 is deprotonated at pH >3 and that it protonates only at low pH, with a pK(a) of 2.3. The M intermediate in the wild-type photocycle accumulates only at high pH, with an apparent pK(a) of 9, via deprotonation of a residue interacting with Asp85, presumably His57. In liposomes reconstituted with ESR, the pK(a) values for M formation and spectral shifts are 2-3 pH units lower than in DDM. The distinctively different pH dependencies of the protonation of Asp85 and the accumulation of the M intermediate in the wild-type protein versus the H57M mutant indicate that there is strong Asp-His interaction, which substantially lowers the pK(a) of Asp85 by stabilizing its deprotonated state.

Control of retinal isomerization in bacteriorhodopsin in the high-intensity regime.

A learning algorithm was used to manipulate optical pulse shapes and optimize retinal isomerization in bacteriorhodopsin, for excitation levels up to 1.8 x 10(16) photons per square centimeter. Below 1/3 the maximum excitation level, the yield was not sensitive to pulse shape. Above this level the learning algorithm found that a Fourier-transform-limited (TL) pulse maximized the 13-cis population. For this optimal pulse the yield increases linearly with intensity well beyond the saturation of the first excited state. To understand these results we performed systematic searches varying the chirp and energy of the pump pulses while monitoring the isomerization yield. The results are interpreted including the influence of 1-photon and multiphoton transitions. The population dynamics in each intermediate conformation and the final branching ratio between the all-trans and 13-cis isomers are modified by changes in the pulse energy and duration.

Shuttling between two protein conformations: the common mechanism for sensory transduction and ion transport.

It has recently become known that light-dependent interconversions between two protein conformations underlie both ion transport in bacteriorhodopsin and halorhodopsin and phototaxis signaling by the sensory rhodopsins of halobacteria. In the transport proteins, the two conformations facilitate alternating access of an occluded ion-binding site to the two surfaces of the membrane, and in the sensory receptors the conformations modulate signal-transducer activity. In sensory rhodopsin I, the same conformational equilibrium is implicated in providing both sensory signaling when bound to its transducer and proton transport when free.

Proton transfer pathways in bacteriorhodopsin at 2.3 angstrom resolution.

Photoisomerization of the retinal of bacteriorhodopsin initiates a cyclic reaction in which a proton is translocated across the membrane. Studies of this protein promise a better understanding of how ion pumps function. Together with a large amount of spectroscopic and mutational data, the atomic structure of bacteriorhodopsin, determined in the last decade at increasing resolutions, has suggested plausible but often contradictory mechanisms. X-ray diffraction of bacteriorhodopsin crystals grown in cubic lipid phase revealed unexpected two-fold symmetries that indicate merohedral twinning along the crystallographic c axis. The structure, refined to 2.3 angstroms taking this twinning into account, is different from earlier models, including that most recently reported. One of the carboxyl oxygen atoms of the proton acceptor Asp85 is connected to the proton donor, the retinal Schiff base, through a hydrogen-bonded water and forms a second hydrogen bond with another water. The other carboxyl oxygen atom of Asp85 accepts a hydrogen bond from Thr89. This structure forms the active site. The nearby Arg82 is the center of a network of numerous hydrogen-bonded residues and an ordered water molecule. This network defines the pathway of the proton from the buried Schiff base to the extracellular surface.

Protein catalysis of the retinal subpicosecond photoisomerization in the primary process of bacteriorhodopsin photosynthesis.

The rate of retinal photoisomerization in wild-type bacteriorhodopsin (wt bR) is compared with that in a number of mutants in which a positively charged (Arg(82)), a negatively charged (Asp(85) or Asp(212)), or neutral hydrogen bonding (Asp(115) or Tyr(185)) amino acid residue known to be functionally important within the retinal cavity is replaced by a neutral, non-hydrogen bonding one. Only the replacements of the charged residues reduced the photoisomerization rate of the 13-cis and all-trans isomers present in these mutants by factors of approximately 1/4 and approximately 1/20, respectively. Retinal photo- and thermal isomerization catalysis and selectivity in wt bR by charged residues is discussed in terms of the known protein structure, the valence-bond wave functions of the ground and excited state of the retinal, and the electrostatic stabilization interactions within the retinal cavity.

Crystal structure of sensory rhodopsin II at 2.4 angstroms: insights into color tuning and transducer interaction.

We report an atomic-resolution structure for a sensory member of the microbial rhodopsin family, the phototaxis receptor sensory rhodopsin II (NpSRII), which mediates blue-light avoidance by the haloarchaeon Natronobacterium pharaonis. The 2.4 angstrom structure reveals features responsible for the 70- to 80-nanometer blue shift of its absorption maximum relative to those of haloarchaeal transport rhodopsins, as well as structural differences due to its sensory, as opposed to transport, function. Multiple factors appear to account for the spectral tuning difference with respect to bacteriorhodopsin: (i) repositioning of the guanidinium group of arginine 72, a residue that interacts with the counterion to the retinylidene protonated Schiff base; (ii) rearrangement of the protein near the retinal ring; and (iii) changes in tilt and slant of the retinal polyene chain. Inspection of the surface topography reveals an exposed polar residue, tyrosine 199, not present in bacteriorhodopsin, in the middle of the membrane bilayer. We propose that this residue interacts with the adjacent helices of the cognate NpSRII transducer NpHtrII.

Structural changes in bacteriorhodopsin during ion transport at 2 angstrom resolution.

Crystal structures of the Asp96 to Asn mutant of the light-driven proton pump bacteriorhodopsin and its M photointermediate produced by illumination at ambient temperature have been determined to 1.8 and 2.0 angstroms resolution, respectively. The trapped photoproduct corresponds to the late M state in the transport cycle-that is, after proton transfer to Asp85 and release of a proton to the extracellular membrane surface, but before reprotonation of the deprotonated retinal Schiff base. Its density map describes displacements of side chains near the retinal induced by its photoisomerization to 13-cis,15-anti and an extensive rearrangement of the three-dimensional network of hydrogen-bonded residues and bound water that accounts for the changed pKa values (where Ka is the acid constant) of the Schiff base and Asp85. The structural changes detected suggest the means for conserving energy at the active site and for ensuring the directionality of proton translocation.

Conversion of bacteriorhodopsin into a chloride ion pump.

In the light-driven proton pump bacteriorhodopsin, proton transfer from the retinal Schiff base to aspartate-85 is the crucial reaction of the transport cycle. In halorhodopsin, a light-driven chloride ion pump, the equivalent of residue 85 is threonine. When aspartate-85 was replaced with threonine, the mutated bacteriorhodopsin became a chloride ion pump when expressed in Halobacterium salinarium and, like halorhodopsin, actively transported chloride ions in the direction opposite from the proton pump. Chloride was bound to it, as revealed by large shifts of the absorption maximum of the chromophore, and its photointermediates included a red-shifted state in the millisecond time domain, with its amplitude and decay rate dependent on chloride concentration. Bacteriorhodopsin and halorhodopsin thus share a common transport mechanism, and the interaction of residue 85 with the retinal Schiff base determines the ionic specificity.

Bacteriorhodopsin.

High-resolution maps from X-ray diffraction of bacteriorhodopsin and some of its photointermediates have yielded insights into how the isomerization of the bound retinal drives ion transport. Although important mechanistic details are still undecided, the events of the photochemical cycle are now understood to reflect changes in specific hydrogen bonds of protein groups and bound water molecules in response to motions of the retinal chain.

Crystallographic studies of the conformational changes that drive directional transmembrane ion movement in bacteriorhodopsin.

Recent advances in the determination of the X-ray crystallographic structures of bacteriorhodopsin, and some of its photointermediates, reveal the nature of the linkage between the relaxation of electrostatic and steric conflicts at the retinal and events elsewhere in the protein. The transport cycle can be now understood in terms of specific and well-described displacements of hydrogen-bonded water, and main-chain and side-chain atoms, that lower the pK(a)s of the proton release group in the extracellular region and Asp-96 in the cytoplasmic region. Thus, local electrostatic conflict of the photoisomerized retinal with Asp-85 and Asp-212 causes deprotonation of the Schiff base, and results in a cascade of events culminating in proton release to the extracellular surface. Local steric conflict of the 13-methyl group with Trp-182 causes, in turn, a cascade of movements in the cytoplasmic region, and results in reprotonation of the Schiff base. Although numerous questions concerning the mechanism of each of these proton (or perhaps hydroxyl ion) transfers remain, the structural results provide a detailed molecular explanation for how the directionality of the ion transfers is determined by the configurational relaxation of the retinal.

Proton translocation mechanism and energetics in the light-driven pump bacteriorhodopsin.

In spite of many still unsolved problems, the mechanism and energetics of the light-driven proton transport are now basically understood. Energy captured during photoexcitation, and retained in the form of bond rotations and strains of the retinal, is transformed into directed changes in the pKa values of vectorially arranged proton transfer groups. The framework for the spatial and temporal organization of these changes is provided by the protein near the retinal Schiff base. The transport is completed by proton transfer among three essential groups in three domains lying roughly parallel with the membrane plane (Fig. 1): (a) the anionic D85 that is included in a complex of residues on the extracellular side containing also R82, D212, Y57 and bound water; (b) the protonated Schiff base; and (c) the protonated D96 that is included in a complex of residues on the cytoplasmic side containing also R227, T46, S226, and bound water. Other neighboring polar groups and water bound elsewhere which play a role in the transport do so either by further influencing the pKa values of the three protonable groups, or by providing passive pathways for proton transfer. The Schiff base proton, destabilized after photoexcitation, is transferred to the low pKa group D85 located on the extracellular side. The access of the deprotonated Schiff base then changes to the cytoplasmic side (the 'reprotonation switch') and its proton affinity increases. Finally, the proton of the high pKa group D96, with access to the cytoplasmic side, is destabilized by a protein conformational change through rearrangement of R227, T46, S226 and bound water, and becomes transferred to the Schiff base. As shown schematically in Fig. 3, these internal events are coupled to proton release and uptake at the two aqueous surfaces. The charge of the extracellular hydrogen-bonded complex is redistributed upon protonation of D85, and if the pH is above the pKa of the complex a proton is released to the bulk. After reprotonation of the Schiff base the pKa of the cytoplasmic hydrogen-bonded complex is raised well above the pH, and D96 regains a proton from the bulk. If the pH is lower than the pKa of the extracellular complex the proton release is delayed until the end of the photocycle. In either sequence there is net transfer of a proton from the cytoplasmic to the extracellular phase. The transfer of excess free energy from the chromophore to the protein, and finally to the transported proton, is described by a characteristic thermodynamic cycle.(ABSTRACT TRUNCATED AT 400 WORDS)

Passive potassium ion permeability of Halobacterium halobium cell envelope membranes.

Cell envelope vesicles, prepared from Halobacterium halobium, were loaded with 3 M KCl, suspended in 3 M NaCl, and the loss of K+ was followed at various temperatures. The Arrhenius plot of the K+-efflux rates shows a break at 30 degrees C, with higher energy of activation above the break. This temperature dependence is consistent with earlier studies of chain motions in liposomes prepared from isolated lipids. The efflux of K+ is more rapid with increasing pH between pH 5 and 7. Since these vesicles do not respire under the experimental conditions it was expected that the K+-efflux data would be related to the passive permeability of the membranes to K+. The apparent K+ permeability at 30 degrees C is 1--2 - 10(-10) cm - s-1. This value corresponds to a 5-h half-life for retained K+ in the envelope vesicles and to a probably much longer half-life in whole cells. The previously observed ability of Halobacterium to retain K+ in the absence of metabolism can thus be explained solely by the permeability characteristics of the membranes.

Current-voltage characteristics of planar lipid membranes with attached Halobacterium cell-envelope vesicles.

We have studied the photoactivity of a system consisting of large, planar, essentially solvent free bilayers bearing adsorbed cell-envelope vesicles prepared from Halobacterium halobium (strain L 33). The system was made conductive by addition of a proton carrier (SF-6847). We observed photocurrents which were linearly dependent upon transmembrane voltage. Current-voltage curves were found to be well described by an equivalent circuit with the following significant parameters: planar bilayer conductance, planar bilayer-vesicle contact area conductance, cell-envelope vesicle conductance, and chloride pump equivalent voltage-generator potential. These parameters are uniquely obtained as a result of a few independent current measurements. The stationary photovoltage was dependent upon chloride concentration, and from this dependence an active transport (pump) affinity of the system for chloride was calculated to be about 50 mM.

Influence of the size and protonation state of acidic residue 85 on the absorption spectrum and photoreaction of the bacteriorhodopsin chromophore.

The consequences of replacing Asp-85 with glutamate in bacteriorhodopsin, as expressed in Halobacterium sp. GRB, were investigated. Similarly to the in vitro mutated and in Escherichia coli expressed protein, the chromophore was found to exist as a mixture of blue (absorption maximum 615 nm) and red (532 nm) forms, depending on the pH. However, we found two widely separated pKa values (about 5.4 and 10.4 without added salt), arguing for two blue and two red forms in separate equilibria. Both blue and red forms of the protein are in the two-dimensional crystalline state. A single pKa, such as in the E. coli expressed protein, was observed only after solubilization with detergent. The photocycle of the blue forms was determined at pH 4.0 with 610 nm photoexcitation, and that of the red forms at pH 10.5 and with 520 nm photoexcitation, in the time-range of 100 ns to 1 s. The blue forms produced no M, but a K- and an L-like intermediate, whose spectra and kinetics resembled those of blue wild-type bacteriorhodopsin below pH 3. The red forms produced a K-like intermediate, as well as M and N. Only the red forms transported protons. Specific perturbation of the neighborhood of the Schiff base by the replacement of Asp-85 with glutamate was suggested by (1) the shift and splitting of the pKa for what is presumably the protonation of residue 85, (2) a 36 nm blue-shift in the absorption of the all-trans red chromophore and a 25 nm red-shift of the 13-cis N chromophore, as compared to wild-type bacteriorhodopsin and its N intermediate, and (3) significant acceleration of the deprotonation of the Schiff base at pH 7, but not of its reprotonation and the following steps in the photocycle.

Evidence of local conformational fluctuations and changes in bacteriorhodopsin, dependent on lipids, detergents and trimeric structure, as studied by 13C NMR.

We examined how the local conformation and dynamics of [3-13C]Ala-labeled bacteriorhodopsin (bR) are altered as viewed from 13C NMR spectra when the natural membrane lipids are partly or completely replaced with detergents. It turned out that the major conformational features of bR, the alphaII-helices, are generally unchanged in the delipidated or solubilized preparations. Upon partial delipidation or detergent solubilization, however, a significant conformational change occurs, ascribed to local conversion of alphaII-->alphaI-helix (one Ala residue involved), evident from the upfield displacement of the transmembrane helical peak from 16.4 ppm to 14.5 ppm, conformational change (one or two Ala residues) within alphaII-helices from 16.4 to 16.0 ppm, and acquired flexibility in the loop region (especially at the F-G loop) as manifested from suppressed peak-intensities in cross-polarization magic angle spinning (CP-MAS) NMR spectra. On the other hand, formation of monomers as solubilized by Triton X-100, Triton N-101 and n-dodecylmaltoside is characterized by the presence of a peak at 15.5 ppm and a shifted absorption maximum (550 nm). The size of micelles under the first two conditions was small enough to yield 13C NMR signals observable by a solution NMR spectrometer, although 13C CP-MAS NMR signals were also visible from a fraction of large-sized micelles. We found that the 16.9 ppm peak (three Ala residues involved), visible by CP-MAS NMR, was displaced upfield when Schiff base was removed by solubilization with sodium dodecyl sulfate, consistent with our previous finding of bleaching to yield bacterioopsin.

Bacteriorhodopsin.

Bacteriorhodopsin is a seven-transmembrane helical protein that contains all-trans retinal. In this light-driven pump, a reaction cycle initiated by photoisomerization to 13-cis causes translocation of a proton across the membrane. Local changes in the geometry of the protonated Schiff base and the proton acceptor Asp85, and the proton conductivities of the half channels that lead from this active site to the two membrane surfaces, interact so as to allow timely proton transfers that result in proton release on the extracellular side and proton uptake on the cytoplasmic one. The details of the steps in this photocycle, and the underlying principles that ensure unidirectionality of the movement of a proton across the protein, provide strong clues to how ion pumps function.

The proton transfers in the cytoplasmic domain of bacteriorhodopsin are facilitated by a cluster of interacting residues.

The stepwise internal proton transfer reactions across the membrane, and the release and uptake at the surface, are the elementary steps that together constitute the transport mechanism in a proton pump. Although the proton donor and acceptor residues can be usually identified, the directionality and the energetics of the proton transfer must be determined to a large extent also by interactions of these with neighboring groups. We have examined the roles of residues D96, T46 and R227 in proton transfers during the photocycle of bacteriorhodopsin near its cytoplasmic surface, and in general the relationship between the reprotonation of the Schiff base and the subsequent proton uptake from the cytoplasmic side. The phenotypes of single and double mutants suggest close functional interaction among D96, T46, R227, and probably internal bound water. Measurements of the free energies of activation indicate that mechanistic interpretation of the rates changed by residue replacements is hindered by a general tendency toward lowered activation enthalpies in the mutated proteins. There is less ambiguity in the free energy levels of the photointermediates. It appears from these that the inhibitory and stimulatory influences of T46 and R227, respectively, on D96 as a proton donor compensate one another and ensure the effective reprotonation of the Schiff base. T46 and D96 mediate, in turn, proton uptake at the cytoplasmic surface. Although ultimately this will reprotonate D96, the observation of proton uptake from the bulk in R82Q without reprotonation of the aspartate residue suggests that the direct proton acceptor is not D96. The results thus indicate that the passage of the proton from the surface to the Schiff base is facilitated by multiple residue and water interactions in the cytoplasmic domain.

Transient channel-opening in bacteriorhodopsin: an EPR study.

Active translocation of ions across membranes requires alternating access of the ion binding site inside the pump to the two membrane surfaces. Proton translocation by bacteriorhodopsin (bR), the light-driven proton pump in Halobacterium salinarium, involves this kind of a change in the accessibility of the centrally located retinal Schiff base. This key event in bR's photocycle ensures that proton release occurs to the extracellular side and proton uptake from the cytoplasmic side. To study the role of protein conformational changes in this reprotonation switch, spin labels were attached to pairs of engineered cysteine residues in the cytoplasmic interhelical loops of bR. Light-induced changes in the distance between a spin label on the EF interhelical loop and a label on either the AB or the CD interhelical loop were observed, and the changes were monitored following photoactivation with time-resolved electron paramagnetic resonance (EPR) spectroscopy. Both distances increase transiently by about 5 A during the photocycle. This opening occurs between proton release and uptake, and may be the conformational switch that changes the accessibility of the retinal Schiff base to the cytoplasmic surface after proton release to the extracellular side.

Light-induced rotation of a transmembrane alpha-helix in bacteriorhodopsin.

Spin labeling EPR spectroscopy has been used to characterize light-induced conformational changes of bacteriorhodopsin (bR). Pairs of nitroxide spin labels were attached to engineered cysteine residues at strategic positions near the cytoplasmic ends of transmembrane alpha-helices B, F, and G in order to monitor distance changes upon light activation. The EPR analysis of six doubly labeled bR mutants indicates that the cytoplasmic end of helix F not only tilts outwards, but also rotates counter-clockwise during the photocycle. The direction of the rotation of helix F is the opposite of the clockwise rotation previously reported for bovine rhodopsin. The opposite chirality of the F helix rotation in the two systems is perhaps related to the differences in the cis-trans photoisomerization of the retinal in the two proteins. Using time-resolved EPR, we monitored the rotation of helix F also in real time, and found that the signal from the rotation arises concurrently with the reprotonation of the retinal Schiff base.