Evolution in the laboratory: the genome of Halobacterium salinarum strain R1 compared to that of strain NRC-1.

We report the sequence of the Halobacterium salinarum strain R1 chromosome and its four megaplasmids. Our set of protein-coding genes is supported by extensive proteomic and sequence homology data. The structures of the plasmids, which show three large-scale duplications (adding up to 100 kb), were unequivocally confirmed by cosmid analysis. The chromosome of strain R1 is completely colinear and virtually identical to that of strain NRC-1. Correlation of the plasmid sequences revealed 210 kb of sequence that occurs only in strain R1. The remaining 350 kb shows virtual sequence identity in the two strains. Nevertheless, the number and overall structure of the plasmids are largely incompatible. Also, 20% of the protein sequences differ despite the near identity at the DNA sequence level. Finally, we report genome-wide mobility data for insertion sequences from which we conclude that strains R1 and NRC-1 originate from the same natural isolate. This exemplifies evolution in the laboratory.

Two pumps, one principle: light-driven ion transport in halobacteria.

Comparison of the primary structure of the chloride pump halorhodopsin with that of the proton pump bacteriorhodopsin provides insight into light-driven ion transport by retinal proteins. Several conserved amino acid residues in the membrane-spanning region of both proteins and their interaction with different isomerization states of retinal are suggested to be the key element for ion transport in both proteins.

Chloride- and pH-dependent proton transport by BR mutant D85N.

Photocurrents from purple membrane suspensions of D85N BR mutant adsorbed to planar lipid membranes (BLM) were recorded under yellow (lambda > 515 nm), blue (360 nm < lambda < 420 nm) and white (lambda > 360 nm) light. The pH dependence of the transient and stationary currents was studied in the range from 4.5 to 10.5. The outwardly directed stationary currents in yellow and blue light indicate the presence of a proton pumping activity, dependent on the pH of the sample, in the same direction as in the wild-type. The inwardly directed currents in white light, due to an inverse proton translocation, in a two-photon process, show a pH dependence as well. The stationary currents in blue and white light are drastically increased in the presence of azide, but not in yellow light. The concentration dependence of the currents on azide indicates binding of azide to the protein. In the presence of 1 M sodium chloride, the stationary proton currents in yellow light show an increase by a factor of 25 at pH 5.5. On addition of 50 mM azide, the stationary current in yellow light decreases again, possibly by competition between azide and chloride for a common binding site. The observed transport modes are discussed in the framework of the recently published IST model for ion translocation by retinal proteins [U. Haupts et al., Biochemistry 36 (1997) 2-7].

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.

Chloride and proton transport in bacteriorhodopsin mutant D85T: different modes of ion translocation in a retinal protein.

Replacement of aspartate 85 (D85) in bacteriorhodopsin (BR) by threonine but not be asparagine creates at pH<7 an anion-binding site in the molecular similar to that in chloride pump halorhodopsin. Binding of various anions to BR-D85T causes a blue shift of the absorption maximum by maximally 57 nm. Connected to this color change is a change in the absorption difference spectrum of the initial state and the longest living photo intermediate from a positive difference maximum at 460 nm in the absence of transported anions to one at 630 nm in their presence. Increasing anion concentration cause decreasing decay times of this intermediate. At physiological pH, BR-D85T but not BR-D85N transports chloride ions inward in green light, protons outward in blue or green light and protons inward in white light (directions refer to the intact cell). The proton movements are observable also in BR-D85N. Thus, creation of an anion-binding site in BR is responsible for chloride transport and introduction of anion-dependent spectroscopic properties at physiological pH. The different transport modes are explained with the help of the recently proposed IST model, which states that after light-induced isomerization of the retinal an ion transfer step and an accessibility change of the active site follow. The latter two steps occur independently. In order to complete the cyclic event, the accessibility change, ion transfer and isomerization state have to be reversed. The relative rates of accessibility changes and ion transfer steps define ultimately the vectoriality of ion transfers. All transport modes described here for the same molecule can satisfactorily be described in the framework of this general concept.

Proton translocation by bacteriorhodopsin in the absence of substantial conformational changes.

Unlike wild-type bacteriorhodopsin (BR), the BR triple mutant D96G/F171C/F219L has been shown to undergo only minor structural rearrangements during its photocycle. Nonetheless, the mutant is capable of transporting protons at a rate of 125(+/-40) H+/BR per minute under light-saturating conditions. Light adaptation of the triple mutant's retinal proceeds in a pH-dependent manner up to a maximum of 63% all-trans. These two findings imply that the transport activity of the triple mutant comprises 66% of the wild-type activity. Time-resolved spectroscopy reveals that the identity and sequence of intermediates in the photocycle of the triple mutant in the all-trans configuration correspond to that of wild-type BR. The only differences relate to a slower rise and decay of the M and O intermediates, and a significant spectral contribution from a 13-cis component. No indication for accumulation of the N intermediate is found under a variety of conditions that normally favor the formation of this species in wild-type BR. The Fourier transform infrared (FTIR) spectrum of the M intermediate in the triple mutant resembles that of wild type. Minor changes in the amide I region during the photocycle suggest that only small movements of the protein backbone occur. Electron microscopy reveals large differences in conformation between the unilluminated state of the mutant protein and wild-type but no light-induced changes in time-resolved measurements. Evidently, proton transport by the triple mutant does not require the major conformational rearrangements that occur on the same time-scale with wild-type. Thus, we conclude that large conformational changes observed in the photocycle of the wild-type and many BR mutants are not a prerequisite for the change in accessibility of the Schiff base nitrogen atom that must occur during vectorial catalysis to allow proton transport.

Protein conformational changes in the bacteriorhodopsin photocycle.

We report a comprehensive electron crystallographic analysis of conformational changes in the photocycle of wild-type bacteriorhodopsin and in a variety of mutant proteins with kinetic defects in the photocycle. Specific intermediates that accumulate in the late stages of the photocycle of wild-type bacteriorhodopsin, the single mutants D38R, D96N, D96G, T46V, L93A and F219L, and the triple mutant D96G/F171C/F219L were trapped by freezing two-dimensional crystals in liquid ethane at varying times after illumination with a light flash. Electron diffraction patterns recorded from these crystals were used to construct projection difference Fourier maps at 3.5 A resolution to define light-driven changes in protein conformation. Our experiments demonstrate that in wild-type bacteriorhodopsin, a large protein conformational change occurs within approximately 1 ms after illumination. Analysis of structural changes in wild-type and mutant bacteriorhodopsins under conditions when either the M or the N intermediate is preferentially accumulated reveals that there are only small differences in structure between M and N intermediates trapped in the same protein. However, a considerably larger variation is observed when the same optical intermediate is trapped in different mutants. In some of the mutants, a partial conformational change is present even prior to illumination, with additional changes occurring upon illumination. Selected mutations, such as those in the D96G/F171C/F219L triple mutant, can sufficiently destabilize the wild-type structure to generate almost the full extent of the conformational change in the dark, with minimal additional light-induced changes. We conclude that the differences in structural changes observed in mutants that display long-lived M, N or O intermediates are best described as variations of one fundamental type of conformational change, rather than representing structural changes that are unique to the optical intermediate that is accumulated. Our observations thus support a simplified view of the photocycle of wild-type bacteriorhodopsin in which the structures of the initial state and the early intermediates (K, L and M1) are well approximated by one protein conformation, while the structures of the later intermediates (M2, N and O) are well approximated by the other protein conformation. We propose that in wild-type bacteriorhodopsin and in most mutants, this conformational change between the M1 and M2 states is likely to make an important contribution towards efficiently switching proton accessibility of the Schiff base from the extracellular side to the cytoplasmic side of the membrane.

Bacteriorhodopsin mutants D85N, D85T and D85,96N as proton pumps.

Proton translocation in the BR mutants D85N, D85T and D85,96N was studied by attachment of purple membranes to planar lipid bilayers. Pump currents in these mutants were measured via capacitive coupling and by use of the appropriate ionophores. All mutants have a reduced pK of their Schiff bases around 8-8.5 in common. At physiological pH, a mixture of chromophores absorbing at 410 nm (deprotonated form) and around 600 nm (protonated form) coexists. Excitation with continuous blue light induces in all three mutants an outwardly directed stationary pump current. These currents are enhanced upon addition of azide in D85N and D85,96N by a factor of 50, but no azide enhancement is observed in D85T. Yellow light alone induces transient inwardly directed currents in the mutants but additional blue light leads to a stationary current with the same direction. All the observed currents are carried by protons, so that the consecutive absorption of a yellow and a blue photon leads to inverted stationary photocurrents by the mutants, as observed with halorhodopsin (HR). A mechanistic model describing the inversion of proton pumping is discussed by the cis-trans, trans-cis isomerization of the retinal and the different proton accessibility of the Schiff base from the extracellular or the cytoplasmic side of the membrane.

The photocycle of the chloride pump halorhodopsin. II: Quantum yields and a kinetic model.

The quantum yield of the primary photoreaction of HR478 was determined as phi(green) = 0.34 +/- 0.02 and that of the photochemical reconversion of HRL410 into HR578 as phi(blue) = 0.01 +/- 0.004. From steady-state illumination and flash-photolysis experiments, a quantitative description of HRL410 formation and decay was made leading to a model of the photocycle in isolated, light-adapted HR. This model satisfies all known facts about HR including its photochronic behaviour.

A unifying concept for ion translocation by retinal proteins.

First, halorhodopsin is capable of pumping protons after illumination with green and blue light in the same direction as chloride. Second, mutated bacteriorhodopsin where the proton acceptor Asp85 and the proton donor Asp96 are replaced by Asn showed proton pump activity after illumination with blue light in the same direction as wildtype after green light illumination. These results can be explained by and are discussed in light of our new hypothesis: structural changes in either molecule lead to a change in ion affinity and accessibility for determining the vectoriality of the transport through the two proteins.

Bacteriorhodopsin can function without a covalent linkage between retinal and protein.

Light energy is transferred from retinal to the protein in bacteriorhodopsin after absorption of a photon resulting in changes of protein conformation. To examine whether the covalent bond, formed by the carbonyl group of retinal and the epsilon-amino group of lysine 216, is essential for this process, a mutant with lysine 216 replaced by alanine was expressed in Halobacterium salinarium L33 (BO-, retinal+). Reconstitution of the chromoprotein with varying retinylidene-n-alkylamines was possible in isolated membranes as well as in whole cells. When the protein in membranes with retinylidene Schiff bases of n-alkylamines of different lengths was reconstituted, the most stable chromoprotein was formed with retinylideneethylamine. The absorbance maximum was at 475 nm in alkaline solution and 620 nm in acidic solution. At neutral pH values both species equilibrate with a third one absorbing maximally at 568 nm. Reconstitution of whole cells with retinylideneethylamine led to a specific proton pump activity of 30 mol of protons per mol of BR per minute. This value indicates a lower limit of transport; no light saturation could be reached in these measurements in contrast to wild-type BR where transport activities of 162 mol of protons per mol of BR per minute under identical conditions can be achieved. Action spectra from flash photolysis experiments revealed that only the 568-nm form led to a M-intermediate with a half-time of decay of 17 ms. In summary, it could be shown that the covalent linkage between retinal and the protein is basically not required for the function of bacteriorhodopsin as a light-driven proton pump.

Closing in on bacteriorhodopsin: progress in understanding the molecule.

Bacteriorhodopsin is the best understood ion transport protein and has become a paradigm for membrane proteins in general and transporters in particular. Models up to 2.5 A resolution of bacteriorhodopsin's structure have been published during the last three years and are basic for understanding its function. Thus one focus of this review is to summarize and to compare these models in detail. Another focus is to follow the protein through its catalytic cycle in summarizing more recent developments. We focus on literature published since 1995; a comprehensive series of reviews was published in 1995 (112).

Roles of cytoplasmic arginine and threonine in chloride transport by the bacteriorhodopsin mutant D85T.

In the light-driven anion pump halorhodopsin (HR), the residues arginine 200 and threonine 203 are involved in anion release at the cytoplasmic side of the membrane. Because of large sequence homology and great structural similarities between HR and bacteriorhodopsin (BR), it has been suggested that anion translocation by HR and by the chloride-pumping BR mutant BR-D85T occurs by the same mechanism. Consequently, the functions of the R200/T203 pair in HR should be the same as those of the corresponding pair in BR-D85T (R175/T178). We have put this hypothesis to a test by creating two mutants of BR-D85T in which R175 and T178 were replaced by glutamine and valine, respectively. Chloride transport activities were essentially the same for all three mutants, whereas chloride binding and the kinetics of parts of the photocycle were markedly affected by the replacement of T178. In contrast, the consequences of mutating R175 proved to be less significant. These findings are consistent with evidence obtained on HR and therefore support the idea that the respective mechanistic roles of the cytoplasmic arginine/threonine pairs in HR and BR-D85T are equal.

Light-driven proton or chloride pumping by halorhodopsin.

Halorhodopsin from Halobacterium halobium was purified and reconstituted with lipids from purple membranes. The resulting protein-containing membrane sheets were adsorbed to a planar lipid membrane and photoelectric properties were analyzed. Depending on light conditions, halorhodopsin acted either as a light-driven chloride pump or as a proton pump: green light caused chloride transport and additional blue light induced proton pumping. In the living cell, both to these vectorial processes would be directed toward the cytoplasm and, compared to ion transport by bacteriorhodopsin, this is an inversed proton flow. Azide, a catalyst for reversible deprotonation of halorhodopsin, enhanced proton transport, and the deprotonated Schiff base in the 13-cis configuration (H410) was identified as the key intermediate of this alternative catalytic cycle in halorhodopsin. While chloride transport in halorhodopsin is mediated by a one-photon process, proton transport requires the absorption of two photons: one photon for formation of H410 and release of a proton, and one photon for photoisomerization of H410 and re-formation of H578 with concomitant uptake of a proton by the Schiff base.

Electrogenic Cl- pump in Acetabularia.

Measurements of this transmembrane potential difference (V) under various conditions have demonstrated the operation of an electrogenic Cl- pump in the outer plasma membrane (plasmalemma) of the unicellular marine alga Acetabularia. In preparations of partly purified membranes (containing plasmalemma), there is Cl- stimulated, N,N'-dicyclohexylcarbodiimide-insensitive, vanadate-sensitive ATPase activity with a pH optimum around pH 6.5. These properties are consistent with the assumption that the electrogenic Cl- pump is an ATPase. In order to investigate electrical details of the "Mitchellian" type of charge-translocating enzyme, steady-state current-voltage curves of the electrogenic pump (Ip(V)) were measured in vivo under dark and light conditions and analysed by two-state reaction kinetic model. This model with the resulting parameters predicts V-sensitive, undirectional Cl- effluxes through the pump. The predictions of this model agree with the experimental results. Green light causes a fast decrease of V, which is explained as a disturbance of the pump cycle. Relaxation studies on this effect and reaction kinetic analysis of Ip(V) under different external Cl- concentrations are used to develop a consistent three-state model of the pump that includes the order of and absolute rate constants of individual reactions, states of charge, stoichiometry, voltage-sensitivity and density of the pump molecules in the membrane.

Reconstitution of bacteriorhodopsin from the apoprotein and retinal studied by Fourier-transform infrared spectroscopy.

The reconstitution of the retinal-containing protein bacteriorhodopsin (BR) from the apoprotein and retinal has been studied by Fourier-transform infrared (FTIR) difference spectroscopy. 9-cis-Retinal which occupies the binding site but does not reconstitute the chromophore was used as "caged retinal". Photoisomerization to the all-trans isomer triggers the reconstitution reaction. Absorption bands in the FTIR difference spectra of the educt and product of the reaction could be assigned by comparison with a 9-cis-retinal FTIR spectrum or an FT-Raman spectrum of BR and due to band shifts observed upon deuterium exchange. Specific difference bands were assigned to the protonated carboxyl groups of D96 and D115 by use of the mutants D115N and D96N. Both aspartic acids are protonated also in the apoprotein with pKa values above 10 and undergo a frequency shift toward higher wavenumbers indicating a more hydrophobic environment in the reconstituted protein. No indication was found for protonation changes of carboxyl groups or other protonatable residues when carrying out the reaction at pH values between 4 and 10. The pH-dependent protonation changes reported earlier [Fischer & Oesterhelt (1980) Biophys. J. 31, 139-146] therefore may be caused by protons in a hydrogen-bonded network. Mutations of E204, but not of D38 or E9, cancel proton uptake during reconstitution at high pH as well as proton release at low pH. It is concluded, that E204, without changing its protonation state itself, is part of a protonatable hydrogen-bonded network which changes its pKa during reconstitution thereby causing the observed protonation changes.

Experimental evidence for hydrogen-bonded network proton transfer in bacteriorhodopsin shown by Fourier-transform infrared spectroscopy using azide as catalyst.

Experimental evidence for proton transfer via a hydrogen-bonded network in a membrane protein is presented. Bacteriorhodopsin's proton transfer mechanism on the proton uptake pathway between Asp-96 and the Schiff base in the M-to-N transition was determined. The slowdown of this transfer by removal of the proton donor in the Asp-96-->Asn mutant can be accelerated again by addition of small weak acid anions such as azide. Fourier-transform infrared experiments show in the Asp-96-->Asn mutant a transient protonation of azide bound to the protein in the M-to-N transition and, due to the addition of azide, restoration of the IR continuum band changes as seen in wild-type bR during proton pumping. The continuum band changes indicate fast proton transfer on the uptake pathway in a hydrogen-bonded network for wild-type bR and the Asp-96-->Asn mutant with azide. Since azide is able to catalyze proton transfer steps also in several kinetically defective bR mutants and in other membrane proteins, our finding might point to a general element of proton transfer mechanisms in proteins.

General concept for ion translocation by halobacterial retinal proteins: the isomerization/switch/transfer (IST) model.

Bacteriorhodopsin (BR), which transports protons out of the cell in a light-driven process, is one of the best-studied energy-transducing proteins. However, a consensus on the exact molecular mechanism has not been reached. Matters are complicated by two experimental facts. First, recent results using BR mutants (BR-D85T) and the homologous protein sensory rhodopsin I demonstrate that the vectoriality of active proton transport may be reversed under appropriate conditions. Second, in BR-D85T as well as in the homologous halorhodopsin, protons and chloride ions compete for transport; e.g. the same molecule may transport either a positive or a negative ion. To rationalize these results, we propose a general model for ion translocation by bacterial rhodopsins which is mainly based on two assumptions. First, the isomerization state of the retinylidene moiety governs the accessibility of the Schiff base in the protein; e.g. all-trans, 15-anti, and 13-cis-15-anti direct the Schiff base to extracellular and cytoplasmic accessibility, respectively, but change in accessibility (called the "switch") is a time-dependent process in the millisecond time range. A light-induced change of the isomerization state induces not only a change in accessibility but also an ion transfer reaction. Second, we propose that these two processes are kinetically independent, e.g. that relative rate constants in a given molecule determine which process occurs first, ultimately defining the vectoriality of active transport.

Inversion of proton translocation in bacteriorhodopsin mutants D85N, D85T, and D85,96N.

Proton translocation activity of bacteriorhodopsin mutants lacking the proton acceptor Asp-85 was investigated using the black lipid membrane technique. Mutants D85N, D85T, and D85,96N were constructed and homologously expressed in Halobacterium salinarium to yield a membrane fraction with a buoyant density of 1.18 g/cm3, i.e., identical to that of wild-type purple membrane. In all mutants, the absorbance maximum was red-shifted between 27 and 49 nm compared with wild type, and the pKa values of the respective Schiff bases were reduced to between 8.3 and 8.9 compared with the value of > 13 in wild type. Therefore, a mixture of chromophores absorbing at 410 nm (deprotonated form) and around 600 nm (protonated form) exists at physiological pH. In continuous blue light, the deprotonated form generates stationary photocurrents. The currents are enhanced by a factor of up to 50 upon addition of azide in D85N and D85,96N mutants, whereas D85T shows no azide effect. The direction of these currents is the same as in wild type in yellow light. Yellow light alone is not sufficient to generate stationary currents in the mutants, but increasing yellow light intensity in the presence of blue light leads to an inversion of the current. Because all currents are carried by protons, this two-photon process demonstrates an inverted proton translocation by BR mutants.

Impedance of the electrogenic Cl(-) pump inAcetabularia: Electrical frequency entrainements, voltage-sensitivity, and reaction kinetic interpretation.

Reaction kinetic analysis of the electrical properties of the electrogenic Cl(-) pump inAcetabularia has been extended from steady-state to nonsteady-state conditions: electrical frequency responses of theAcetabularia membrane have been measured over the range from 1 Hz to 10 kHz at transmembrane potential differences across the plasmalemma (V m ) between -70 and -240 mV using voltage-clamp techniques. The results are well described by an electrical equivalent circuit with three parallel limbs: a conventional membrane capacitancec m , a steadystate conductanceg o (predominantly of the pump pathway plus a minor passive ion conductance) and a conductanceg s in series with a capacitancec p which are peculiar to the temporal behavior of the pump. The absolute values and voltage sensitivities of these four elements have been determined:c m of about 8 mF m(-2) turned out to be voltage insensitive; it is considered to be normal.g o is voltage sensitive and displays a peak of about 80 S m(-2) around -180 mV. Voltage sensitivity ofg s could not be documented due to large scatter ofg s (around 80 S m(-2)).c p behaved voltage sensitive with a notch of about 20 mF m(-2) around -180 mV, a peak of about 40 mF m(-2) at -120 mV and vanishing at -70 mV. When these data are compared with the predictions of nonsteady-state electrical properties of charge transport systems (U.-P. Hansen, J. Tittor, D. Gradmann, 1983,J. Membrane Biol. in press), model "A" (redistribution of states within the reaction cycle) consistently provides magnitude and voltage sensitivity of the elementsg o ,g s andc p of the equivalent circuit, when known kinetic parameters of the pump are used for the calculations. This analysis results in a density of pump elements in theAcetabularia plasmalemma of about 50 nmol m(-2). The dominating rate constants for the redistribution of the individual states of the pump in the electric field turn out to be in the range of 500 sec(-1), under normal conditions.