ESR, a light-driven proton pump from Exiguobacterium sibiricum, contains a lysine residue (Lys96) in the proton donor site. Substitution of Lys96 with a nonionizable residue greatly slows ...reprotonation of the retinal Schiff base. The recent study of electrogenicity of the K96A mutant revealed that overall efficiency of proton transport is decreased in the mutant due to back reactions (Siletsky et al., BBA, 2019). Similar to members of the proteorhodopsin and xanthorhodopsin families, in ESR the primary proton acceptor from the Schiff base, Asp85, closely interacts with His57. To examine the role of His57 in the efficiency of proton translocation by ESR, we studied the effects of H57N and H57N/K96A mutations on the pH dependence of light-induced pH changes in suspensions of Escherichia coli cells, kinetics of absorption changes and electrogenic proton transfer reactions during the photocycle. We found that at low pH (<5) the proton pumping efficiency of the H57N mutant in E. coli cells and its electrogenic efficiency in proteoliposomes is substantially higher than in the WT, suggesting that interaction of His57 with Asp85 sets the low pH limit for H+ pumping in ESR. The electrogenic components that correspond to proton uptake were strongly accelerated at low pH in the mutant indicating that Lys96 functions as a very efficient proton donor at low pH. In the H57N/K96A mutant, a higher H+ pumping efficiency compared with K96A was observed especially at high pH, apparently from eliminating back reactions between Asp85 and the Schiff base by the H57N mutation.
•ESR is a proton pump with Lys96 at the site of the H+ donor to the Schiff base (SB).•His57 near H+ acceptor (Asp85) controls proton affinity of the latter.•At low pH, the H57N mutant pumps protons more efficiently than the WT.•H57N mutation eliminates/decreases back reactions between Asp85 and the SB.•Lys96 acts as a fast and efficient donor at neutral and low pH.
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 pK a ∼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 pK a ∼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 pK a ∼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.
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.
•ESR is a proton pump with an unusual H+ donor to the Schiff base (SB), Lys96.•Asp and Glu effectively replace Lys as H+ donors in the K96D/A47T and K96E mutants.•Reprotonation of the SB is pH-dependent in ESR and its mutants.•The donors in ESR connect to the SB and the bulk in the M → N transition.•The carboxylic H+ donors are reprotonated quickly after transferring H+ to SB in ESR.
ESR from Exiguobacterium sibiricum is a retinal protein which functions as a proton pump. Unusual feature of ESR is that a lysine residue is present at a site for the internal proton donor, which in ...other proton pumps is a carboxylic residue. Replacement of Lys96 with alanine slows reprotonation of the Schiff base by two orders of magnitude, indicating that Lys96 and interacting water molecules function as internal proton donor to the Schiff base. In this work we examined time resolved generation of light-induced electric potential ΔΨ by the K96A mutant reconstituted into proteoliposomes. We found that the ΔΨ component, which accompanied reprotonation of the Schiff base in wild type ESR, was not only slowed but also decreased greatly in the mutant, and negative phase appeared at high pH. This indicates a higher probability of back reactions in ESR than in bacteriorhodopsin since no negative components have been observed in homologous mutants of BR, D96N and D96A. The higher rate of back reactions in ESR is probably caused by different arrangement of the proton acceptor site compared to that in BR and different sequence of proton release and uptake. Addition of sodium azide, which substitutes for the internal proton donor, restores both the rate and amplitude of the ΔΨ components related to the Schiff base reprotonation in the K96A mutant. This indicates that overall proton transport results from competition of forward and reverse reactions, and emphasizes the importance of internal donor for high efficiency and directionality of H+ transfer.
•ESR is a proton pump with Lys at the site of the H+ donor to the Schiff base (SB).•K96A mutation slows reprotonation of the SB by two orders of magnitude.•Electrogenecity of the SB reprotonation is reduced in K96A mutant; azide restores it.•This indicates presence of reverse reactions which decrease directionality of the pump.•Fast SB reprotonation is important for high efficiency of H+ transport in ESR.
Overproduction of the membrane proteins in
Escherichia coli
cells is a common approach to obtain sufficient material for their functional and structural studies. However, the efficiency of this ...process can be limited by toxic effects which decrease the viability of the host and lead to low yield of the product. During the expression of the esterase autotransporter AT877 from
Psychrobacter cryohalolentis
K5
T
, we observed significant growth inhibition of the C41(DE3) cells in comparison with the same cells producing other recombinant proteins. Induction of AT877 synthesis also resulted in the elevated expression of a magnesium transporter MgtA and decreased ATP content of the cells. To characterize the response to overexpression of the autotransporter in bacterial cells, we performed a comparative analysis of their proteomic profile by mass spectrometry. According to the obtained data,
E. coli
cells which synthesize AT877 experience complex stress condition presumably associated with secretion apparatus overloading and improper localization of the recombinant protein. Several response pathways were shown to be activated by AT877 overproduction including Cpx, PhoP/PhoQ, Psp, and σ
E
The obtained results open new opportunities for optimization of the recombinant membrane protein expression in
E. coli
for structural studies and biotechnological applications.
Photochromic ultrafast reactions of bacteriorhodopsin (H. salinarum) and bovine rhodopsin were conducted with a femtosecond two-pump probe pulse setup with the time resolution of 20–25fs. The ...dynamics of the forward and reverse photochemical reactions for both retinal-containing proteins was compared. It is demonstrated that when retinal-containing proteins are excited by femtosecond pulses, dynamics pattern of the vibrational coherent wave packets in the course of the reaction is different for bacteriorhodopsin and visual rhodopsin. As shown in these studies, the low-frequencies that form a wave packets experimentally observed in the dynamics of primary products formation as a result of retinal photoisomerization have different intensities and are clearer for bovine rhodopsin. Photo-reversible reactions for both retinal proteins were performed from the stage of the relatively stable photointermediates that appear within 3–5ps after the light pulse impact. It is demonstrated that the efficiency of the reverse phototransition K-form→bacteriorhodopsin is almost five-fold higher than that of the Batho-intermediate→visual rhodopsin phototransition. The results obtained indicate that in the course of evolution the intramolecular mechanism of the chromophore-protein interaction in visual rhodopsin becomes more perfect and specific. The decrease in the probability of the reverse chromophore photoisomerization (all-trans→11-cis retinal) in primary photo-induced rhodopsin products causes an increase in the efficiency of the photoreception process.
•Comparative study of bacteriorhodopsin and bovine rhodopsin photochromic reactions.•A femtosecond two-pump probe pulse setup with the time resolution of 20–25fs.•Photo-reversible reaction of bacteriorhodopsin from the K-intermediate stage.•Photo-reversible reaction of bovine rhodopsin from the Batho-intermediate stage.•The efficiency of reverse photo-transition for bovine rhodopsin is five-fold lower.
The autotransporter AT877 from
Psychrobacter cryohalolentis
belongs to the family of outer membrane proteins containing N-terminal passenger and C-terminal translocator domains that form the basis ...for the design of display systems on the surface of bacterial cells. It was shown in our previous study that the passenger domain of AT877 can be replaced by the cold-active esterase EstPc or the tenth domain of fibronectin type III (
10
Fn3). In order to increase efficiency of the
10
Fn3 surface display in
Escherichia coli
cells, four deletion variants of the Fn877 hybrid autotransporter were obtained. It was demonstrated that all variants are present in the membrane of bacterial cells and facilitate binding of the antibodies specific against
10
Fn3 on the cell surface. The highest level of binding is provided by the variants Δ239 and Δ310, containing four and seven beta-strands out of twelve that comprise the structure of the translocator domain. Using electrophoresis under semi-native conditions, presence of heat modifiability in the full-size Fn877 and its deletion variants was demonstrated, which indicated preservation of beta structure in their molecules. The obtained results could be used to optimize the bacterial display systems of
10
Fn3, as well as of other heterologous passenger domains.
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.
A retinal protein from Exiguobacterium sibiricum (ESR) functions as a light-driven proton pump. Unlike other proton pumps, it contains Lys96 instead of a usual carboxylic residue in the internal ...proton donor site. Nevertheless, the reprotonation of the Schiff base occurs fast, indicating that Lys96 facilitates proton transfer from the bulk. In this study we examined kinetics of light-induced transmembrane electrical potential difference, ΔΨ, generated in proteoliposomes reconstituted with ESR. We show that total magnitude of ΔΨ is comparable to that produced by bacteriorhodopsin but its kinetic components and their pH dependence are substantially different. The results are in agreement with the earlier finding that proton uptake precedes reprotonation of the Schiff base in ESR, suggesting that Lys96 is unprotonated in the initial state and gains a proton transiently in the photocycle. The electrogenic phases and the photocycle transitions related to proton transfer from the bulk to the Schiff base are pH dependent. At neutral pH, they occur with τ 0.5ms and 4.5ms. At alkaline pH, the fast component ceases and Schiff base reprotonation slows. At pH8.4, a spectrally silent electrogenic component with τ 0.25ms is detected, which can be attributed to proton transfer from the bulk to Lys96. At pH5.1, the amplitude of ΔΨ decreases 10 fold, reflecting a decreased yield and rate of proton transfer, apparently from protonation of the acceptor (Asp85-His57 pair) in the initial state. The features of the photoelectric potential generation correlate with the ESR structure and proposed mechanism of proton transfer.
•ESR is a H+ pump with Lys in the position of the H+ donor to the retinal Schiff base.•The kinetics of light-induced membrane potential generation by ESR was examined.•The rates and the electrogenic distances of proton transfer steps were obtained.•They indicate different mechanism of H+ uptake and release in ESR, compared to BR.
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.
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