Insight into a single halobacterium using a dual-bacteriorhodopsin system with different functionally optimized pH ranges to cope with periplasmic pH changes associated with continuous light illumination
Department of Biochemical Science and Technology, College of Life Science, National Taiwan University, Taipei, Taiwan
The light-driven outward proton transporter assists energy production via an ATP synthase system best exemplified by the bacteriorhodopsin (BR) from Halobacterium salinarum, HsBR. As the only archaea able to survive in the resource-limited ecosystem of the Dead Sea, Haloarcula marismortui has been reported to have a unique dual-BR system, consisting of HmBRI and HmBRII, instead of only a single BR in a cell (solo-BR). The contribution of this dual-BR system to survival was investigated. First, native H. marismortui and H. salinarum cells were tested in water that had been adjusted to mimic the conditions of Dead Sea water. These archaea were shown to accumulate protons and reduce pH in their periplasmic regions, which disabled further proton transportation functionality in H. salinarum but not in H. marismortui. Then, pH-dependent photocurrent measurements using purified BR proteins demonstrated that HsBR and HmBRI were functional at pH > 5.0 and that HmBRII was functional at pH > 4.0. Our results indicate that the dual-HmBR system is composed of two BRs with different optimal functional pH ranges and together they maintain light-driven proton transport activity under pH > 4.0, which might contribute the survival of H. marismortui under the acidic pH of the Dead Sea.
The Dead Sea is one of the most saline lakes on earth, with salinity nearing 340 g l−1. Only a few varieties of microorganisms have been found to be able to survive in this resource-limited environment, including red halophilic archaea (Haloarcula marismortui), unicellular green algae (Dunaliella parva Lerche), and various types of bacteria, protozoa and filamentous fungi (Wilkansky, 1936; Buchalo et al., 1998; Jin et al., 2005). As the Dead Sea lacks a complete food chain such as those observed in other marine or terrestrial ecosystems, solar light harvesting has become the most prevalent and reliable pathway mediating energy harvest and conversion.
Solar light was also the main energy source in the resource-limited, early global environment, approximately 3.5 billion years ago (Schopf, 2006), allowing the development of proteins and protein complexes with small light-sensitive chemical compounds, termed chromophores, which directly link photon capture to photosensing and photoenergy harvesting in a diverse range of microbial organisms and plants (Briggs and Spudich, 2005). One of these photoreceptors is microbial rhodopsin, which features a seven-alpha-helix transmembrane region with a chromophore, identified as all-trans retinal that covalently binds to a lysine residue in the seventh helix. Microbial rhodopsin belongs to a diverse group of photoactive proteins found in organisms of the Archaea, Bacteria and Eukarya domains (Spudich, 2006).
Among the archaea, energy capture is mediated by a superfamily of light-driven proton transporters, bacteriorhodopsins (BRs). BRs have been intensively studied over nearly four decades, using HsBR from Halobacterium salinarum, and the light-triggered outward proton releasing mechanism is well understood (Oesterhelt and Stoeckenius, 1971; Lanyi, 2004). HsBR is encoded by the bop gene (Ng et al., 2000) and expressed in the purple membrane of H. salinarum cells; it cooperates with ATP synthases to produce ATP, a biologically consumable form of energy.
One area of interest regarding BRs is their ability to maintain functionality under persistent and intense sunlight, a biological condition shared by almost all halobacteria, including those in the Dead Sea. The protons released via continuously light-driven BRs have been shown to roam and accumulate around the lipid-periplasmic region (Antoranz Contera et al., 2010). Such an increase in local proton concentration was also hinted at and supported by studies that demonstrated that a BR modified with a pH-sensitive fluorescent probe (Alexiev et al., 1994; Scherrer et al., 1994) had a fluorescence increase upon light activation and the detection of pH imbalance in the juxtamembranous compartments and extracellular space when using BR as a tool for optogenetic studies (Yizhar et al., 2011). A strong acidic environment is not optimal for BRs (Zimanyi et al., 1992), and observing H. marismortui and other solo-BR halobacteria under continuous illumination and exposure to different pH conditions is a logical approach to begin investigating the differences between solo- and dual-BR systems.
Solo-BR systems, which contain only one BR in a cell, are nearly ubiquitous among members of the Class Halobacteria, but H. marismortui was proposed to have an unusual dual-BR system after genome project analyses (Baliga et al., 2004; Bolhuis et al., 2006). Our previous study (Fu et al., 2010) verified that a dual-BR system, containing both HmBRI and HmBRII, indeed exists in the six-rhodopsin system of H. marismortui, making H. marismortui the only dual-BR system identified to date.
As the organism lives in the relatively resource-deprived Dead Sea, the biological significance of such a unique dual-BR system in H. marismortui remains unknown. We have demonstrated that both HmBRI and HmBRII have light-driven proton transporters and share similar action spectra in detergents, but no significant difference in biological, biochemical and biophysical properties have been identified. In addition, whether the system is responsible for H. marismortui being the only archaea that can survive in the Dead Sea has yet to be elucidated.
In this report, H. marismortui and H. salinarum cells, as well as their proteins, including HmBRI, HmBRII, and the well-studied BR from H. salinarum, HsBR (all of them were cloned, expressed and purified using Escherichia coli hetero expression system), were examined with various biophysical assays. It was found that continuous illumination led to a pH decrease in periplasmic regions in both types of archaea and attenuated the light-driven proton release in HsBR and HmBRI but not in HmBRII. The dual-BR system in H. marismortui contains HmBRI and HmBRII, and these two proteins have different optimal pH ranges for functionality, and these ranges appear to complement each other. The biological significance of these ranges is examined and discussed.
pH dependence of light-driven proton transport in native halobacterium cells
To first confirm that pH changes in the periplasmic region occur under light illumination and to elucidate the physiological significance of a dual-BR system, the light-driven proton releasing activity of native halobacterium cells featuring either dual-BR or solo-BR systems was directly measured, with slight modifications from the reference protocol (Sudo and Spudich, 2006). Briefly, as the pH of the Dead Sea is approximately 5.5, and seawater pH is limited to the 7.5–8.4 range (Dickson, 1993; Oren and Gunde-Cimerman, 2012), both types of halobacterium cells were resuspended in solutions with different designated pH values to cover those ranges. In addition, light-driven proton release was estimated from the pH changes measured by probes in the surrounding solutions. The results indicated the H. salinarum cells, which have a solo-BR system (Fig. 1B and C), and H. marismortui cells, which have a dual-BR system (Fig. 1E and F), had comparable light-dependent proton transport patterns at pH 7.0 (Fig. 1B and E) and pH 8.5 (Fig. 1C and F). A major difference was observed at lower pH ranges. The light-driven proton transport activity reached a plateau in H. salinarum (Fig. 1A) cells at pH 5.5, whereas H. marismortui (Fig. 1D) cells presented consistent proton release transportation trends similar to those observed at pH 7.0 and 8.5. One explanation for these physiological results is that at least one HmBR protein in the H. marismortui cells can perform light-driven proton translocation under acidic pH conditions, but the solo-BR system of H. salinarum cells cannot. The fact that light-dependent pH changes were directly detectable supports the notion that pH changes occur in the periplasmic regions of native cells under consistent illumination.
pH dependence of the retinal chromophore absorption band of BRs and its relationship to the protonation of counter ions
With pH changes occurring in the periplasmic regions of illuminated cells, an important rhodopsin feature that required investigation was the pH-dependent maximum absorbance, λmax, or the activation wavelength, which indicate the microenvironment of the retinal chromophore. To test the λmax shift under different pH conditions (Fig. 2), the purified wild-type proteins of HmBRI, HmBRII and HsBR were individually incubated with different buffered solutions of pH 2.0 and 9.0 for 2 h in the dark, and the absorbance spectrum, scanning from 250 to 750 nm, was measured for each sample. An approximately 55 nm redshift in the λmax for HsBR (Fig. 2A) and a redshift of approximately 15 nm in HmBRI (Fig. 2B) were recorded. Surprisingly, no shift was recorded in HmBRII (Fig. 2C); this type of λmax resistance to change at low-pH conditions observed in HmBRII has not been previously reported. These results led to further assays determining the whole-protein pKa and individual BR pH-dependent functionality.
To examine the influence of outside pH on the microenvironment of a Schiff base and proton acceptor, such as Asp85 in HsBR, a whole-protein pH titration measurement, an assay similar to the pKa determination, was performed for both HmBRs and the HsBR from pH 1.5 to pH 10.0 (Fig. 3). Based on the fitting of the spectral transitions of all three BRs, the whole protein pKa values were estimated to be 3.7 for HsBR (Fig. 3A and D) and 2.7 for HmBRI (Fig. 3B and D). Both values are comparable to those obtained in assays of the purple membrane in previous studies (Turner et al., 1993; Imasheva et al., 2006). The whole protein pKa for HmBRII (Fig. 3C); however, could not be determined due to the limited absorbance shift (Fig. 3D), but it can be reasonably estimated to have a pKa of less than 1.6. This unusually low pKa observed for HmBRII is the lowest ever determined for any light-driven proton pump microbial rhodopsin, including those of the proteorhodopsins (PRs) identified from proteobacteria in the ocean with pKa of about 7–7.5 (Kelemen et al., 2003). These results indicate more integrated or less pH-induced microenvironmental changes around the proton acceptor/donor, or Schiff base, and the results of the whole protein pKa measurements suggest that outside pH environments differently affect the light-driven responses of HmBRs. A detailed study at pH < 1.5 was unpractical because the proteins become unstable and precipitation in this pH range.
pH dependence light-induced HmBRI and HmBRII photocurrents
Our previous study demonstrated the light-driven proton transport nature of both recombinant HmBRI and HmBRII in E. coli (Fu et al., 2010). The further pH-dependent light-driven proton transport activity of both purified HmBRs was examined based on a photocurrent measurement assay recently established by Chu et al. (2010). Briefly, the proteins were dissolved in solution with a pH range from 3.0 to 9.0 (Fig. 4), and upon illumination with light, the net movement of protons was measured by the generated current. The signals recorded at pH < 3 were unpractical because the ITO electrode was known to become electrochemical instable under this pH range (Senthilkumar et al., 2008). Therefore, the measurements were conducted under pH 3.0–9.0. HmBRI (Fig. 4B and D) presented an immediate proton-releasing dominant pattern at pH 5.0–9.0, similar to that observed in the purple membrane of HsBR (Fig. 4A and D), and HmBRI also demonstrated light-driven net uptake dominance at pH 3.0–4.0, a phenomenon interpreted as a significant reduction in the light-driven protons releasing from the proton releasing complex (Lanyi, 2006; Chu et al., 2010). However, HmBRII (Fig. 4B and D) appeared to have a light-driven proton-release dominant pattern at pH 4.0–9.0, and inward-uptake dominance was observed at pH values lower than 4.0. These results indicate that the local environmental pH values affected the light-driven proton currents differently in HmBRI and HmBRII.
Effects of the D85N and D96N mutants on the spectroscopic properties of HmBRI and HmBRII
To further investigate the possible molecular mechanisms of the observed pH-dependent differences among the BRs, the neutral counter ion and proton-reuptake mutants were introduced into wild-type HmBRI and HmBRII, which was followed by several biophysical measurements. First, as the typical UV/Vis spectral blue shifts of the absorption maxima are associated with the deprotonation of the aspartate that interacts with the Schiff base. This aspartate is known to serve as a main component of its counter ion and proton acceptor during the photocycle, e.g. Asp85 in HsBR (PDB: 1C3W). A full neutralization of the negative charge on Asp85 was achieved via the construction of a D85N mutant of both HmBRI and HmBRII (D83N-HmBRI and D91N-HmBRII) (Fig. S2). The D85N-HsBR appeared to have the same λmax as that of wild-type HsBR incubated in pH 2 (Fig. 2A dash line). In D83N-HmBRI and D91N-HmBRII, unlike that in HsBR, the mutant pigments exhibited maximum absorption at 580 nm and 565 nm at pH 5.8 respectively (Fig. 2B and C dash line). These proteins presented 28 nm and 13 nm red shifts, respectively, compared with the absorption maximum at pH 5.8, and 41 nm and 15 nm red shifts relative to the maximum absorption at pH 9 in the wild-type HmBRI and HmBRII respectively. The much smaller shift in the HmBRII absorption maximum suggests that only a minor fraction of Asp85 might be protonated even at pH 2.
Second, the pH-dependent absorption maxima changes were determined with point mutations in the critical aspartate for proton re-uptake, corresponding to the Asp96 in HsBR (Lanyi, 2006), of the two HmBRs. Both D96N-corresponding mutants of HmBRI and HmBRII (D94N-HmBRI and D102N-HmBRII) (Fig. S2) demonstrated pH-dependent spectra comparable to wild-type proteins, respectively (Fig. 5A and B), indicating this Asp96 position in both HmBRs was less influenced by outside pH environment changes.
Third, as the kinetics of the photocycle roughly depict and reflect the light-induced proton release and re-uptake efficiency in BR-like proteins, the photocycles (Fig. 5) of D94N-HmBRI and D102N-HmBRII were measured. The ground-state recovery (Fig. 5C and D), the M-intermediate formation and decay (Fig. S3) at different pH levels indicated that the ground-state recovery delay at pH 5.8 (green), comparable to the delay at pH 4.0 (orange), was observed in D94N-HmBRI (Fig. 5C) but not in D102N-HmBRII (Fig. 5D). Both mutant proteins presented a slower ground-state recovery delay at pH 7.8 (Fig. 5C and D, navy). For both wild-type HmBRs (Fig. S4), no significant difference was observed in the pH dependence of ground-state recovery, M-formation or recovery rate.
We previously reported, albeit with no information of biological significance, on the first and only dual-BR system, composed of HmBRI and HmBRII, in H. marismortui, which contains a six-rhodopsin system (Fu et al., 2010). In this report, we found that the two HmBRs in native H. marismortui cells, as opposed to the E. coli-overexpressed proteins, exerted the same light-driven proton release at two different optimal pH ranges and that H. marismortui is capable of maintaining at least one operational BR at a pH ranging from at least 4.0 to 9.0, which allows the organism to handle the environmental pH oscillations in the lipid-periplasmic space.
In the early global environment, BRs might have been beneficial for organism survival, at least for Halobacterium, by functioning as light-driven outward proton transporters that generate proton gradients for the subsequent conversion of solar energy into chemical energy through ATP synthases. H. marismortui is the only member of the Archaea that can survive in the Dead Sea. The H. marismortui genome project revealed HmBRI and HmBRII, which were proposed to absorb maximally at different wavelengths to extend their efficiency for solar energy harvesting (Baliga et al., 2004). Because our previous results demonstrated that they actually both absorbed the same wavelength of light at pH 5.8, suggesting that they both function as light-driven proton transporters (Fu et al., 2010), we investigated their pH-dependent protein functionalities along with H. salinarum, which is known to have a solo-BR system.
The reported proton roaming in the purple membrane of H. salinarum led us to revisit the dual-BR system in H. marismortui. BRs under illumination can potentially accumulate protons. A pH decrease was demonstrated by both fluorescence-modified bacteriorhodopsin (Alexiev et al., 1994; Scherrer et al., 1994) and a study demonstrating the light-driven released protons roaming the lipid-periplasmic region (Antoranz Contera et al., 2010). As the main difference between those two biological systems is that, in addition to having different average pH values in original habitat environments, one has a solo-BR system and the other has a dual-BR system, it was plausible to further examine whether persistent illumination can induce pH changes in the periplasmic regions the BRs face. The illumination-dependent pH changes experiments (Fig. 1B and E) demonstrated the increase of acidity in native H. salinarum and H. marismortui cells in Dead Sea water-mimicking solution, confirming the proton accumulation in the periplasmic region as the light-driven release of protons saturated the lipids binding sites on the surface of the membrane and were released to the environment. While both cell types presented the same consistent light-induced pH decrease at pH 7.0 and 8.5, different trends at pH 5.5 were observed (Fig. 1A and D). Although the slightly increased signal-to-noise ratio made measurement under pH 5.0 impractical, at pH 5.5, the pH for the Dead Sea, the H. salinarum cells reached a plateau after initial pH decrease, a significant phenomenon that was not observed in H. marismortui cells. Such difference at least is not caused by either cell membrane buffering capacity or the proton motive force energy coupling from Na+/H+ antiporter. Rius et al. reported that there is only minor buffering capacity at pH 5.5 under the light illumination and dark condition for the cell membrane of H. salinarum (Rius and Loren, 1996). Although the light-induced proton motive force form the Na+ gradient in Na+/H+ antiporter (Lanyi and MacDonald, 1976), but a later study showed the activity of the Na+/H+ antiporter was independent to the presence of the BR in H. salinarum (Luisi et al., 1980). The pH-dependent light-driven photocurrent generation ability of individual proteins was examined to further explore this difference.
The purified BRs were soaked in a controlled pH environment, and the results indicated that HmBRI and HmBRII demonstrated light-driven proton release from the proton releasing complex at pH ranging from 5.0 to 9.0 and 4.0 to 9.0 respectively (Fig. 4). HmBRII was therefore deemed to be the first BR to have a consistent protonation of the counter ion against outside acidic conditions (Fig. 2), and its functionality was maintained at lower pH conditions than those reported previously for any BR (Zimanyi et al., 1992). HmBRI, on the other hand, underwent λmax shift under acidic conditions and functioned efficiently at pH above 5.0, similar to HsBR. Interestingly, when the results of light-driven pH changes in the periplasmic regions measured in the native cells (Fig. 1) are compared with the photocurrent pH dependence of both HmBRs combined, as is the case in the dual-BR system in H. marismortui, there is a consistent optimal pH range for BR functionality. HmBRI was analysed and shown to function more like HsBR in H. salinarum. According to our results, the existence of HmBRII was the reason why H. marismortui cells, in contrast to H. salinarum cells, still presented high light-driven proton transport in acidic conditions, maintaining its λmax and functionality at a lower pH. This conclusion is supported by the results reported here, and the phenomenon itself consequently compensates for the low pH inactivation of HmBRI.
Furthermore, the pH-dependent protein λmax shifts were measured. The HmBRII demonstrated the most consistent λmax at any pH above 2.5 (Fig. 3C), whereas HmBRI started to undergo redshifts at a pH less than 4.0 (Fig. 3B), which was similar to the well-studied HsBR (Fig. 3A). The whole protein pKa was calculated (Fig. 3D), and HmBRII was found to have an extremely low pKa of less than 1.6, which is lower than pKa of HmBRI and HsBR. HmBRII's extremely low pKa suggests that the chemical environment around the retinal binding pocket can be maintained across a wide range of different pHs. In other words, HmBRII might be able to maintain the same chemical environment that favours proton translocation at an approximate pH 4.0–9.0 range, spanning five orders of proton concentrations, while low-pH environments lower the effectiveness of HmBRI and HsBR.
Therefore, the difference in the pH-dependent λmax changes observed between HmBRI and HmBRII, the differences in pKa for the whole protein against the environment, together with the differences found in the pH-dependent photocurrent measurements demonstrate that the proteins had different functional activities under different pH conditions. This finding is relevant given the alteration of the pH environment of the extracellular membrane surface, which is mediated by the stiff cationic layer that is condensed at the extracellular surface (Antoranz Contera et al., 2010), that was reported in the purple membrane. The advantage of maintaining light-driven proton transport in a wider range of pH environments and the periplasmic regions is that the H. marismortui cells can sustain higher proton motive force for the ATP synthase system under even intense solar illumination than H. salinarum with the BR which might not regain functionality before the protons accumulated in the periplasmic region were reduced, either by consumption by the ATP synthase system or by diffusion to the environment. Considering that H. marismortui cells are exposed to continuous intense solar light in the relatively resource-deprived Dead Sea in comparison with other haloarchaea and given that they swim at least five times slower than H. salinarum (Lin et al., 2010), a photorepellent response to escape an overdose of sunlight – which results in transient proton accumulation in the periplasmic space defined by the membrane surface and S-layer (Albers et al., 2006) and a subsequent decrease in pH – is plausible.
Such differences in pH-dependent properties between HmBRI and HmBRII suggest possible varieties in the microenvironments of residues involved in light-driven proton transportation (Supplementary note, Figs S1 and S2). The possible regions include the proton releasing group, the proton reuptake group, the residues around a Schiff base involved in isomerization and a proton acceptor/donor. The pH-dependent spectrum changes occurred in wild-type HmBRI but not in HmBRII, inspiring further investigations into the microenvironments around the retinal-binding region. Our mutagenesis studies at the corresponding residue D85 of HsBR demonstrated that the maintenance of microenvironments at the Schiff base region was more D85-independent in HmBRII than in HmBRI, as environmental [H+] affected D83 of HmBRI much significantly than it in D91 of HmBRII. In the proton reuptake region represented by D96 of HsBR, results from the experiments with the D96N-corresponding mutants demonstrated pH-dependent spectrum changes occurring in D94N-HmBRI (Fig. 5A) but not in D102N-HmBRII (Fig. 5B). Furthermore, in pH-dependent photocycle measurements, the kinetics of total recovery time (G back to G) (Fig. 5C and D), M-formation (G to M) and M-decay (M to O) (Fig. S3) were separated and determined for these two mutants and compared with the wild-type. In the wild-type, the pH demonstrated no significant influence in both HmBRI and II (Fig. S4), while in the D96N-corresponding mutants, the pH increase to pH 5.8 from 4.0 slowed the kinetics of M-decay in D94N-HmBRI (Fig. 5C) but not for the D102N-HmBRII until the pH increased to 7.8 (Fig. 5D). These results suggest the proton reuptake region in HmBRII is more independent than in HmBRI.
As D85 and D96 are essential for light-driven proton transportation, HmBRII is likely to have more relatively pH-resistant proton reuptake and proton acceptor microenvironments than those in HmBRI. Although an atomic resolution structure of HmBRII, not necessarily needed for HmBRI as it highly resembles HsBR, and well-chosen mutagenesis studies will surely be important steps towards finding the molecular explanation of such pH dependence, the differences in those two important regions under different pH environments are already clearly shown in this study.
A summary illustration (Fig. 6) shows the functional differences of a dual-BR system as compared with a solo-BR system. The proton accumulation around the local lipid-periplasmic interface region will lead to a functionally diminished HmBRI, but it will not affect the functionality of HmBRII, as shown in our results (Figs 1 and 4). HmBRII will still be capable of performing light-driven proton transportation as the sunlight persists, and consequently, ATP generation will continue. The same can be proposed if the local pH around the lipid-periplasmic interface region becomes higher than 7.0 (Fig. 4D); HmBRI, instead of HmBRII, will present normal light-driven proton release to maintain the necessary proton motive force. It is worth mentioning that the optimal growth pH is 7.5 for H. salinarum (Hassanshahian and Mohamadian, 2011), whereas the Dead Sea, where H. marismortui is the lone archaea that can survive (DasSarma and Arora, 2002), has an average pH of 5.5. The results of this report correspond well to those pH values.
In this report, we demonstrated how continuous light activation leads to pH changes in the periplasmic regions in both native H. marismortui and H. salinarum cells. The protein assays revealed a series of properties that are pH-dependent between HmBRI and HmBRII that were consistently supported by a λmax spectrum shift assay, whole protein pKa estimation, light-driven photocurrent, photocycle measurements, and the light-driven pH changes were explained and measured using native cell assay. This dual-BR function may lead to more efficient conversion of consumable biological energy than the solo-BR system, because the dual-BR system maintains at least one functional BR, HmBRII, for solar energy capture across pH approximately 4.0–9.0, whereas the solo-BR system efficiently functions only at a pH above 5.0. Therefore, these results provide information that will further our understanding of the unique dual-BR system found in H. marismortui.
DNA cloning, protein expression and purification
For DNA manipulation, the bop and xop1 genes of H. marismortui were amplified with PCR from the total genomic DNA as described previously (Fu et al., 2010). The bop gene of H. salinarum was also amplified from its genomic DNA with the addition of a NcoI restriction enzyme recognition sequence before the start codon and a XhoI restriction enzyme recognition sequence before the stop codon. The DNA fragment was treated with restriction enzymes and ligated into a NcoI–XhoI treated pET-21d vector. The expressed N- and C-terminal peptide sequences are as follows:
HsBR: 1MVELL … … .AATSD262LEHHHHHH. The site-directed mutagenesis was performed according to the instruction manual from the QuikChangeTM Lightning Site-Directed Mutagenesis Kit. All the protein samples were prepared as described in a previous study (Fu et al., 2010; 2012). This procedure yielded 5–10 mg per litre of culture for purified HmBRI and HmBRII; the yield of purified HsBR was about 2 mg per litre of culture.
Light-driven proton transporter assay
A light-driven ion transporter assay was conducted, with modification, as previously described (Imasheva et al., 2006). Briefly, both H. marismortui and H. salinarum (a kind gift from Dr Wailap Victor Ng) were grown at 42°C in medium of the following composition (g l−1): NaCl, 250; MgSO4·7H2O, 20; Na3C6H5O7, 3; KCl, 2; peptone (Oxoid, LP0034), 10. The late log phase H. marismortui or H. salinarum cells were pelleted and washed twice with a solution containing 4.28 M NaCl, 81 mM MgSO4 and 27 mM KCl at room temperature. The cells were then suspended in this solution and adjusted to an OD600 of approximately 1 in the dark. The initial pH of all samples was adjusted to corresponding designated conditions. Both halobacterium samples were illuminated with a 1 W continuous LED green laser (532 nm). The pH was monitored in real-time using a pH electrode (Eutech Instruments Cyberscan 2100), which was connected to a computer via an RS232C cable.
A slight modification of the electrochemical cell designed by Chu et al. (2010) was used. The photocurrent measurements were carried out by using a modulated CW 532 nm green laser as the excitation light source. The CW 532 nm green laser was controlled by DAQ with an 850 ms illumination duration. For each measurement, 64 rounds were averaged. The purified HmBR proteins were dialysed against a solution containing 10 mM NaCl, 0.02% n-dodecyl- beta-d-maltoside (DDM), for at least 48 h, and then the proteins were adjusted to the appropriate pH value using 10 mM HCl or NaOH, with real-time monitoring, using a pH meter. The reference cell solution was prepared from the dialysis solution, and the pH value was adjusted using the same strategy as with the protein sample.
The absorption spectroscopy and pH titration of BRs were measured with UV/Vis spectroscopy as described previously (Wang et al., 2003; Sudo and Spudich, 2006; Fu et al., 2010; 2013). Briefly, the purified and concentrated BRs were diluted 100-fold into different pH buffers (KCl, citric acid, phosphate, Tris or NaHCO3, each at 100 mM concentration) under red dim light before absorption spectra measurements were taken (U1900 UV/Vis, Hitachi, Japan). The results were then used to evaluate the pH adaptation action spectrum (λmax) and further pKa determination of the counter ion of the Schiff base. The differential absorbance changes were calculated and plotted against the pH values. The data sets were fitted to a sigmoid curve function to calculate pKa. All measurements were performed at 25°C.
Flash-induced absorption transients
Flash-induced absorption transients were monitored with a flash-photolysis system designed by our group and described previously (Fu et al., 2010). The flash laser was a Nd-YAG laser (532 nm, 6 ns pulse, 40 mJ). The purified proteins were suspended in buffers with different pH conditions to reach 0.3 at ODλmax, and the transient absorbance changes were recorded at selected wavelengths. The curves represent the loss and recovery of absorbance at the indicated wavelength upon green laser (532 nm) excitation. Transients were collected and averaged for each measurement. All measurements were performed at 25°C.
We thank Li-Kang Chu, PhD and Chia-Ling Kuo from the Department of Chemistry, National Tsing-Hua University, Hsin-Chu, Taiwan for assistance with the photocurrent system set-up and preliminary data collection. We are grateful to Prof. John L. Spudich from Center for Membrane Biology, University of Texas Health Science Center at Houston for important suggestions and careful reading of this manuscript. We also thank Simon H.-S. Yeh for his carefully reading of the supplementary information and Kang-cheng Liu and Ching-Che Huang for their assistance with light-driven proton transport assay hardware system set-up. This work was supported by the Yen Tjing Ling Industrial Research Institute, National Taiwan University, Grants 100-S-A71 and sponsored by the Giant Lion Know-How, Taipei, Taiwan.