Xanthorhodopsin (XR) from Salinibacter ruber is a light-driven proton pump containing retinal and a light-harvesting carotenoid antenna salinixanthin. Previous structure-functional studies of XR were conducted using a protein isolated from the native host only due to the absence of heterologous expression in Escherichia coli. In this paper, we describe cell-free synthesis and incorporation in lipid–protein nanodiscs of the recombinant XR that demonstrated its principal compatibility with E. coli biosynthetic machinery. To produce XR in E. coli, three C-terminal deletion variants of this protein were constructed. In contrast to the full-length XR, their expression resulted in efficient synthesis in E. coli cells. However, cells producing recombinant XR variants bound retinal only upon growth in minimal medium, not in the rich one. The XR3 variant with deletion of ten C-terminal amino acid residues was obtained and characterized. Its absorption spectrum and photocycle kinetics were close to those reported for XR isolated from S. ruber membranes and bleached from salinixanthin. We have also constructed the first mutants of XR, H62M and D96N, and examined their properties.

Light-driven proton transport by microbial retinal proteins such as archaeal bacteriorhodopsin involves carboxylic residues as internal proton donors to the catalytic center which is a retinal Schiff base (SB). The proton donor, Asp96 in bacteriorhodopsin, supplies a proton to the transiently deprotonated Schiff base during the photochemical cycle. Subsequent proton uptake resets the protonated state of the donor. This two step process became a distinctive signature of retinal based proton pumps. Similar steps are observed also in many natural variants of bacterial proteorhodopsins and xanthorhodopsins where glutamic acid residues serve as a proton donor. Recently, however, an exception to this rule was found. A retinal protein from Exiguobacterium sibiricum, ESR, contains a Lys residue in place of Asp or Glu, which facilitates proton transfer from the bulk to the SB. Lys96 can be functionally replaced with the more common donor residues, Asp or Glu. Proton transfer to the SB in the mutants containing these replacements (K96E and K96D/A47T) is much faster than in the proteins lacking the proton donor (K96A and similar mutants), and in the case of K96D/A47T, comparable with that in the wild type, indicating that carboxylic residues can replace Lys96 as proton donors in ESR. We show here that there are important differences in the functioning of these residues in ESR from the way Asp96 functions in bacteriorhodopsin. Reprotonation of the SB and proton uptake from the bulk occur almost simultaneously during the M to N transition (as in the wild type ESR at neutral pH), whereas in bacteriorhodopsin these two steps are well separated in time and occur during the M to N and N to O transitions, respectively.

In the last grant period we explored the Na+ binding site of the recently discovered light-driven sodium ion pump. The rationale was that comparison of this novel system to the similar proton pumps and chloride ion pumps would reveal the amazingly (and unexpectedly) wide variety of structural features that govern conversion of light-energy into biologically useful transmembrane gradients and thus production of biomass. A thorough description of this system would establish the basis for continuing our funded research on these proteins.

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 pK a 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.

Microbial rhodopsins function as light-driven (retinal-based) ion pumps, cation channels, or light sensors in various microorganisms [see Heberle et al.'s Retinal Proteins-You can teach an old dog new tricks (1)], are found in all three domains of life (2-4),

A group of microbial retinal proteins most closely related to the proton pump xanthorhodopsin has a novel sequence motif and a novel function. Instead of, or in addition to, proton transport, they perform light-driven sodium ion transport, as reported for one representative of this group (KR2) from Krokinobacter. In this paper, we examine a similar protein, GLR from Gillisia limnaea, expressed in Escherichia coli, which shares some properties with KR2 but transports only Na+. The absorption spectrum of GLR is insensitive to Na+ at concentrations of ≤3 M. However, very low concentrations of Na+ cause profound differences in the decay and rise time of photocycle intermediates, consistent with a switch from a “Na+-independent” to a “Na+-dependent” photocycle (or photocycle branch) at ∼60 μM Na+. The rates of photocycle steps in the latter, but not the former, are linearly dependent on Na+ concentration. This suggests that a high-affinity Na+ binding site is created transiently after photoexcitation, and entry of Na+ from the bulk to this site redirects the course of events in the remainder of the cycle. A greater concentration of Na+ is needed for switching the reaction path at lower pH. The data suggest therefore competition between H+ and Na+ to determine the two alternative pathways. The idea that a Na+ binding site can be created at the Schiff base counterion is supported by the finding that upon perturbation of this region in the D251E mutant, Na+ binds without photoexcitation. Binding of Na+ to the mutant shifts the chromophore maximum to the red like that of H+, which occurs in the photocycle of the wild type.

Significance Archaerhodopsin-3 (Arch) is an integral membrane protein that can function as a genetically encoded fluorescent indicator of membrane voltage in neurons. The ability to visualize changes in membrane voltage is of great interest as a readout for neuronal activity. Published variants of this protein, however, are too dim to enable wide-field imaging of cell populations. We used directed evolution to increase the absolute brightness of Arch as a reporter for optogenetics research and live-cell imaging. This study establishes that introducing mutations around the retinal Schiff-base linkage and screening for increased fluorescence is an effective strategy for generating bright rhodopsin variants. At least some mutations discovered in one rhodopsin ( Gloeobacter violaceus rhodopsin) can be transferred to another (Arch) to increase fluorescence.

The photocycle of the retinal protein from Exiguobacterium sibiricum, which differs from bacteriorhodopsin in both its primary donor and acceptor, is characterized by visible and infrared spectroscopy. At pH above pKa ~6.5, we find a bacteriorhodopsin-like photocycle, which originates from excitation of the all-trans retinal chromophore with K-, L-, M-, and N-like intermediates. At pH below pKa ~6.5, the M state, which reflects Schiff base deprotonation during proton pumping, is not accumulated. However, using the infrared band at ~1760 cm(-1) as a marker for transient protonation of the primary acceptor, we find that Schiff base deprotonation must have occurred at pH not only above but also below the pKa ~6.5. Thus, the M state is formed but not accumulated for kinetic reasons. Further, chromophore reisomerization from the 13-cis to the all-trans conformation occurs very late in the photocycle. The strongly red-shifted states that dominate the second half of the cycle are produced before the reisomerization step, and by this criterion, they are not O-like but rather N-like states. The assignment of photocycle intermediates enables reevaluation of the photocycle; its specific features are discussed in relation to the general mechanism of proton transport in retinal proteins.

A lysine instead of the usual carboxyl group is in place of the internal proton donor to the retinal Schiff base in the light-driven proton pump of Exiguobacterium sibiricum (ESR). The involvement of this lysine in proton transfer is indicated by the finding that its substitution with alanine or other residues slows reprotonation of the Schiff base (decay of the M intermediate) by more than 2 orders of magnitude. In these mutants, the rate constant of the M decay linearly decreases with a decrease in proton concentration, as expected if reprotonation is limited by the uptake of a proton from the bulk. In wild type ESR, M decay is biphasic, and the rate constants are nearly pH-independent between pH6 and 9. Proton uptake occurs after M formation but before M decay, which is especially evident in D2O and at high pH. Proton uptake is biphasic; the amplitude of the fast phase decreases with a pK(a) of 8.5 +/- 0.3, which reflects the pKa of the donor during proton uptake. Similarly, the fraction of the faster component of M decay decreases and the slower one increases, with a pK(a) of 8.1 +/- 0.2. The data therefore suggest that the reprotonation of the Schiff base in ESR is preceded by transient protonation of an initially unprotonated donor, which is probably the is an element of-amino group of Lys-96 or a water molecule in its vicinity, and it facilitates proton delivery from the bulk to the reaction center of the protein.

A lysine instead of the usual carboxyl group is in place of the internal proton donor to the retinal Schiff base in the light-driven proton pump of Exiguobacterium sibiricum (ESR). The involvement of this lysine in proton transfer is indicated by the finding that its substitution with alanine or other residues slows reprotonation of the Schiff base (decay of the M intermediate) by more than 2 orders of magnitude. In these mutants, the rate constant of the M decay linearly decreases with a decrease in proton concentration, as expected if reprotonation is limited by the uptake of a proton from the bulk. In wild type ESR, M decay is biphasic, and the rate constants are nearly pH-independent between pH 6 and 9. Proton uptake occurs after M formation but before M decay, which is especially evident in D2O and at high pH. Proton uptake is biphasic; the amplitude of the fast phase decreases with a pKa of 8.5 ± 0.3, which reflects the pKa of the donor during proton uptake. Similarly, the fraction of the faster component of M decay decreases and the slower one increases, with a pKa of 8.1 ± 0.2. The data therefore suggest that the reprotonation of the Schiff base in ESR is preceded by transient protonation of an initially unprotonated donor, which is probably the ϵ-amino group of Lys-96 or a water molecule in its vicinity, and it facilitates proton delivery from the bulk to the reaction center of the protein.Background: Lysine rather than a carboxylic residue is in place of the internal proton donor in the E. sibiricum proton pump.Results: H+ uptake precedes reprotonation of the retinal Schiff base. K96A mutation slows it by >100-fold.Conclusion: Lysine 96 facilitates proton delivery to the Schiff base.Significance: This is the first example where lysine mediates proton transfer to the retinal Schiff base. A lysine instead of the usual carboxyl group is in place of the internal proton donor to the retinal Schiff base in the light-driven proton pump of Exiguobacterium sibiricum (ESR). The involvement of this lysine in proton transfer is indicated by the finding that its substitution with alanine or other residues slows reprotonation of the Schiff base (decay of the M intermediate) by more than 2 orders of magnitude. In these mutants, the rate constant of the M decay linearly decreases with a decrease in proton concentration, as expected if reprotonation is limited by the uptake of a proton from the bulk. In wild type ESR, M decay is biphasic, and the rate constants are nearly pH-independent between pH 6 and 9. Proton uptake occurs after M formation but before M decay, which is especially evident in D2O and at high pH. Proton uptake is biphasic; the amplitude of the fast phase decreases with a pKa of 8.5 ± 0.3, which reflects the pKa of the donor during proton uptake. Similarly, the fraction of the faster component of M decay decreases and the slower one increases, with a pKa of 8.1 ± 0.2. The data therefore suggest that the reprotonation of the Schiff base in ESR is preceded by transient protonation of an initially unprotonated donor, which is probably the ϵ-amino group of Lys-96 or a water molecule in its vicinity, and it facilitates proton delivery from the bulk to the reaction center of the protein. Background: Lysine rather than a carboxylic residue is in place of the internal proton donor in the E. sibiricum proton pump. Results: H+ uptake precedes reprotonation of the retinal Schiff base. K96A mutation slows it by >100-fold. Conclusion: Lysine 96 facilitates proton delivery to the Schiff base. Significance: This is the first example where lysine mediates proton transfer to the retinal Schiff base. An Unusual Lysine Acts Like a Proton Donor in a Bacterial Proton Pump♦: Breaking the Carboxyl Rule. Lysine 96 Facilitates Reprotonation of the Schiff Base in the Photocycle of a Retinal Protein from Journal of Biological ChemistryVol. 288Issue 29Preview♦ See referenced article, J. Biol. Chem. 2013, 288, 21254–21265 Full-Text PDF Open Access

Xanthorhodopsin is a light-driven proton pump like bacteriorhodopsin, but contains a second chromophore, salinixanthin, a carotenoid. Action spectra for transport and fluorescence of the retinal upon excitation of salinixanthin, as well as femtosecond kinetics of carotenoid fluorescence, indicate that the carotenoid functions as an antenna to the retinal, with ca. 50% efficiency for excited-state energy transfer. The resulting charge redistribution in the retinal and the local electric field produced, in the excited state and in the primary stable photoproduct K, in turn, causes distinct electrochromic shifts in the carotenoid spectrum. The close interaction of carotenoid and retinal suggests that the two chromophores are in proximity. Tight binding of the carotenoid, as indicated by its sharpened vibration bands and intense induced circular dichroism in the visible, is removed in the absence of the retinal, and restored upon reconstitution with retinal or retinal analogues. The crystallographic structure of the xanthorhodopsin at 1.9 Å resolution identifies the location of the chromophores. The structural model from x-ray diffraction reveals that the ring moieties of the retinal and the carotenoid are at nearly van der Waals distance from one another. Salinixanthin and one of the native carotenoids of Gloeobacter, echinenone, bind to gloeorhodopsin expressed in E. coli, and the complex shows excited-state energy exchange much like xanthorhodopsin. Protein sequences and mutational studies in Gloeobacter indicate that binding of the carotenoid depends on a conserved glycine at the keto-ring, which is a tryptophan in bacteriorhodopsin and other archaeal rhodopsins. On this basis, numerous otherwise unrelated eubacterial rhodopsins are predicted to contain light-harvesting carotenoids.

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.

Xanthorhodopsin (xR), a light-driven proton pump, is unique among the rhodopsin family because it contains not one, but two strongly interacting chromophores, the retinal and a carotenoid. The carotenoid is bound to the protein and acts as a light-harvesting antenna, transferring energy to the retinal with ∼40% quantum efficiency (Balashov et al., Science309, 2005). Unlike photosynthetic complexes in which excitation energy transfer (EET) occurs from a large collection of antennas to a reaction center, the EET in xR occurs directly between the retinal and a single bound carotenoid. Thus, xR serves as a simple (and computationally accessible, albeit challenging) model system for understanding EET in light-harvesting systems. Here, we present the results of long (aggregate length > 350 ns) classical molecular dynamics (MD) simulations, starting from the x-ray crystal structure of xR (Luecke et al., PNAS 105, 2008), coupled with single-point quantum mechanical (DFT) calculations performed on hundreds of simulation snapshots, to address two points. First, because the resolution of the crystal structure is not sufficiently high to resolve protons, the protonation state of the counterion complex (specifically, the His62-Asp96 pair) is unknown, so we ran four MD simulations in parallel, each with a different combination of protonation states for the His-Asp pair. The MD simulations, as well as DFT calculations of the retinal excited state energies, identify a single state that is most consistent with experimental data. Second, we probe the effects of small structural changes, which occur within the context of a thermally disordered lipid (POPC) bilayer, on the retinal excitation energies, and we conclude that the environment could play an important role in the EET between the bound carotenoid and the retinal.

: Xanthorhodopsin, a proton pump from the eubacterium Salinibacter ruber, is a unique dual chromophore system that contains, in addition to retinal, the carotenoid salinixanthin as a light-harvesting antenna. The key factors affecting binding and function of the antenna were established and examined. The first is the binding of the carotenoid ring near the retinal ring. Substitution of the small glycine with bulky tryptophan in this site eliminates binding. The second factor is the 4-keto group in the carotenoid ring. A close analogue of salinixanthin, salinixanthol, in which the 4-keto group (C=O) is reduced to hydroxyl (C-OH), does not bind. Third, the binding site itself. Another protein capable of carotenoid binding was found. Gloeobacter rhodopsin, was shown to bind salinixanthin and echinenone but not beta-carotene or salinixanthol. Using femtosecond absorption spectroscopy, a large transient electrochromic shift of the salinixanthin band was detected 150 fs after excitation of the retinal chromophore. It indicates that excitation of retinal is accompanied by charge separation and a strong electrostatic field. It partially remains in the first photoproduct K. Thermal conversion of the latter involves at least two substates which were characterized with FTIR spectroscopy. The kinetics of photocycle and proton transport by xanthorhodopsin was examined.

In the photocycle of bacteriorhodopsin at pH 7, a proton is ejected to the extracellular medium during the protonation of Asp-85 upon formation of the M intermediate. The group that releases the ejected proton does not become reprotonated until the prephotolysis state is restored from the N and O intermediates. In contrast, at acidic pH, this proton release group remains protonated to the end of the cycle. Time-resolved Fourier transform infrared measurements obtained at pH 5 and 7 were fitted to obtain spectra of kinetic intermediates, from which the spectra of M and N/O versus unphotolyzed state were calculated. Vibrational features that appear in both M and N/O spectra at pH 7, but not at pH 5, are attributable to deprotonation from the proton release group and resulting structural alterations. Our results agree with the earlier conclusion that this group is a protonated internal water cluster, and provide a stronger experimental basis for this assignment. A decrease in local polarity at the N-C bond of the side chain of Lys-216 resulting from deprotonation of this water cluster may be responsible for the increase in the proton affinity of Asp-85 through M and N/O, which is crucial for maintaining the directionality of proton pumping.

We report molecular dynamics simulations of the trends in the changes in secondary structure of the seven individual helices of bacteriorhodopsin when inserted into sodium dodecyl sulfate (SDS) micelles, and their dependence on the amino acid sequence. The results indicate that the partitioning of the helices in the micelles and their stability are dependent on the hydrophobicity of the transmembrane segments. Helices A, B, and E are stable and retain their initial secondary structure throughout the 100 ns simulation time. In contrast, helices C, D, F, and G show structural perturbations within the first 10 ns. The instabilities are localized near charged residues within the transmembrane segments. The overall structural instability of the helix is correlated with its partitioning to the surface of the micelle and its interaction with polar groups there. The in silico experiments were performed to complement the in vitro experiments that examined the partial denaturation of bacteriorhodopsin in SDS described in the preceding article (DOI 10.1021/bi201769z). The simulations are consistent with the trends revealed by the experimental results but strongly underestimate the extent of helix to extended coil transformation. The reason may be either that the sampling time was not sufficiently long or, more interestingly, that interhelix residue interactions play a role in the unfolding of the helices.

We characterized the structure of partially unfolded bacteriorhodopsin in sodium dodecyl sulfate (SDS) micelles and compared it with its in vitro refolded structure after reconstitution with dimyristoylphosphatidylcholine/3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (DMPC/CHAPS). Intrahelical and interhelical distances were mapped in the protein using strategically located spin-label pairs at helical ends, assayed by pulsed electron paramagnetic resonance spectroscopy (double electron-electron spin resonance, DEER). We find that in SDS the intrahelical end-to-end distances exhibit broad distributions, suggesting a heterogeneous ensemble of conformations with differing secondary structures. Nevertheless, a majority of the denatured population retains end-to-end distances similar to those in the native state. In contrast, the observed greatly increased interhelical distances, in addition to their very broad distributions, suggest that in the SDS micelles very little of the native tertiary structure remains.

We present a comparative study of xanthorhodopsin, a proton pump with the carotenoid salinixanthin serving as an antenna, and the closely related bacteriorhodopsin. Upon excitation of retinal, xanthorhodopsin exhibits a wavy transient absorption pattern in the region between 470 and 540nm. We interpret this signal as due to electrochromic effect of the transient electric field of excited retinal on salinixanthin. The spectral shift decreases during the retinal dynamics through the ultrafast part of the photocycle. Differences in dynamics of bacteriorhodopsin and xanthorhodopsin are discussed.

Salinixanthin, a C40-carotenoid acyl glycoside, serves as a light-harvesting antenna in the retinal-based proton pump xanthorhodopsin of Salinibacter ruber. In the crystallographic structure of this protein, the conjugated chain of salinixanthin is located at the protein–lipid boundary and interacts with residues of helices E and F. Its ring, with a 4-keto group, is rotated relative to the plane of the π-system of the carotenoid polyene chain and immobilized in a binding site near the β-ionone retinal ring. We show here that the carotenoid can be removed by oxidation with ammonium persulfate, with little effect on the other chromophore, retinal. The characteristic CD bands attributed to bound salinixanthin are now absent. The kinetics of the photocycle is only slightly perturbed, showing a 1.5-fold decrease in the overall turnover rate. The carotenoid-free protein can be reconstituted with salinixanthin extracted from the cell membrane of S. ruber. Reconstitution is accompanied by restoration of the characteristic vibronic structure of the absorption spectrum of the antenna carotenoid, its chirality, and the excited-state energy transfer to the retinal. Minor modification of salinixanthin, by reducing the carbonyl C=O double bond in the ring to a C-OH, suppresses its binding to the protein and eliminates the antenna function. This indicates that the presence of the 4-keto group is critical for carotenoid binding and efficient energy transfer.