Spectroscopic Characterization of Halorhodopsin Reconstituted into Nanodisks Using Native Lipids

Abstract

We successfully reconstituted single Natronomonas pharaonis halorhodopsin (NpHR) trimers into a nanodisk (ND) using the native archaeal lipid (NL) and an artificial lipid having a zwitterionic headgroup, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Incorporation of single trimeric NpHR into NDs was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, size-exclusion chromatography, and visible circular dichroism spectroscopy. The Cl⁻ binding affinity of NpHR in NDs using NL (NL-ND NpHR) or POPC (POPC-ND NpHR) was examined by absorption spectroscopy, showing that the Cl⁻-releasing affinities (K_d,N↔O) of these ND-reconstituted NpHRs are more than 10 times higher than that obtained from native NpHR membrane fragments (MFs) harvested from a NpHR-overexpressing archaeal strain (MF NpHR). The photoreaction kinetics of these ND-reconstituted NpHRs revealed that the Cl⁻ uptake was faster than that of MF NpHR. These differences in the Cl⁻-releasing and uptake properties of ND-reconstituted NpHRs and MF NpHR may arise from suppression of protein conformational changes associated with Cl⁻ release from the trimeric NpHR caused by ND reconstitution, conformational perturbation in the trimeric state, and loss of the trimer-trimer interactions. On the other hand, POPC-ND NpHR demonstrated accelerated Cl⁻ uptake compared to NL-ND NpHR, suggesting that the negative charge on the archaeal membrane surface regulates the photocycle of NpHR. Although NL-ND NpHR and MF NpHR are embedded in the same lipid, the lower Cl⁻-binding affinity at the initial state (K_d,initial) and faster recovering from the NpHR′ state to the original state of the photoreaction cycle were observed for NL-ND NpHR, probably because of insufficient interactions with a chromophore in the native membrane, bacterioruberin in reconstituted NDs. Our results indicate that specific interactions of NpHR with surrounding lipids and bacterioruberin, structural flexibility of the membrane, and interactions between trimeric NpHRs may be necessary for efficient Cl⁻ pumping.

Significance

Natronomonas pharaonis halorhodopsin (NpHR), a cytoplasmic membrane Cl⁻ pump, was reconstituted into nanodisks (NDs) using endogenous and exogenous membranes. The photoreaction cycle was examined by absorption, circular dichroism spectra, and flash photolysis. Based on the thermodynamic and kinetic parameters of the photoreaction in detergent-solubilized NpHRs, NpHRs reconstituted into artificial lipid NDs, and NpHR reconstituted into native membrane NDs, we propose that the functional differences in these NpHRs are due to 1) the low content of bacterioruberin in the membrane, 2) the different charge on the membrane surface, 3) the suppression of the conformational changes associated with the Cl⁻ release, 4) the conformational perturbation in the NpHR trimers, and 5) the lack of the intertrimer interactions of NpHR.

Introduction

Cells of all living organisms are encased in lipid bilayer membranes and contain various intracellular bilayer membranes (e.g., microsomes, organelles, etc.). Transmembrane proteins serve physiologically important functions such as the transport of substances across bilayers or signal transduction, which lead to downstream cellular responses. Therefore, these membrane proteins have been targets for countless intensive studies.

Studying membrane proteins is difficult because of the substantial challenges that plague sample preparations, particularly during the protein purification process. In most cases, detergents are used to solubilize membrane components and extract the membrane proteins from the lipid bilayer while preventing protein aggregation in the aqueous solution. When successful, solubilized membrane proteins are clear in detergent-containing solutions, making them suitable for spectroscopic measurements; however, they are prone to denaturation and inactivity because of the nonphysiological environment. Thus, the structures and functions of the detergent-solubilized proteins are often significantly different from their native state. To maintain the properly folded and active states of membrane proteins, purified proteins can be reconstituted into a mimic of the biological lipid bilayer membrane. However, under these conditions, the turbidity of the membrane suspension results in spectroscopic scattering, which does not allow for quantitative and precise spectroscopic analysis.

Recently, nanodisks (NDs) have been recognized as a useful model membrane system for the study of membrane proteins (1). NDs consist of phospholipids and membrane scaffold proteins (MSPs) derived from apolipoprotein A-I. MSPs form amphipathic α-helical belts that wrap around the phospholipids and allow for the control of ND size by the insertion or deletion of helices in the MSPs. NDs are stable, uniformly sized, and do not aggregate, and thus are clear in solution. These ND characteristics enable the precise spectroscopic measurement of active membrane proteins embedded in lipid membranes under the physiological conditions.

Microbial rhodopsin is a typical integral membrane protein that can be reconstituted into NDs to examine its function under physiological conditions (2). Microbial rhodopsin is a group of transmembrane photoreceptor proteins that contain the apoprotein opsin, have a seven transmembrane α-helical architecture, and have a chromophore (all-trans retinal) that binds to a conserved lysine residue in the seventh transmembrane helix via a protonated Schiff base linkage (3). The microbial rhodopsins are divided into two major classes, ion pumps and signal receptors. Common proton (H⁺) pumps include bacteriorhodopsin (BR) (4) and proteorhodopsin (PR) (5), which pump H⁺ out of the cell in response to light and are found in halophilic Archaea and marine Proteobacteria, respectively. In halophilic Archaea, light stimulates halorhodopsin (HR) pump activity (6), driving Cl⁻ into the cells from the environment. The ion-pumping species of rhodopsin create proton motive forces and inside-negative membrane potentials that drive the production of adenosine triphosphate (ATP) (7). The other class of rhodopsins are the photosensors, represented by sensory rhodopsin I (SRI) (8,9) and II (SRII, also called phoborhodopsin) (9,10), which regulate attractant (SR1) and repellent (SR2) phototaxis responses in halophilic Archaea.

Among them, BR from a halophilic archaeon Halobacterium salinarum (HsBR) (11), green-light absorbing PR (GPR) (12), and SRII from the halo-alkalophilic archaeon Natronomonas pharaonis (NpSRII) (13) were successfully reconstituted into NDs of different sizes and phospholipid compositions. HsBRs were incorporated into ND of ∼9–12 nm in diameter consisting of 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (11). The incorporated HsBR formed a monomer or a trimer, which was confirmed by the size-exclusion chromatography and visible circular dichroism (CD) spectroscopy. The photochemical properties of ND-reconstituted HsBRs were found to be almost the same as those observed for HsBRs in liposomes (11).

More recent studies revealed that the lipid composition between zwitterionic and negatively charged lipids can affect the photoreaction kinetics of HsBR (14). Although the tertiary structure of HsBR did not depend on the lipid composition, the reduced content of the negatively charged lipids in the ND significantly retarded the photocycle kinetics and decreased the population of transient intermediates during photoreaction. Such lipid-dependent photoreaction kinetics were also encountered for GPR (12). The lifetimes of the intermediates in the photocycle of GPR reconstituted with longer lipids and unsaturated acyl chains drastically shortened the intermediate’s lifetimes and the overall photocycle. These results indicate that the composition of the lipid bilayer is a vital component of the mechanism controlling the photophysical properties of GPRs. Additional evidence from native bacterial membranes demonstrated that minor components such as squalene and glycolipid sulfate can significantly alter the decay of the photoreaction intermediates (15). Thus, it is nearly impossible to completely reproduce the native protein-lipid interactions using simple artificial lipids in NDs; however, these alterations can be used to efficiently identify additional mechanistic properties in proteins of interest.

In this study, we reconstituted an archaeal Cl⁻ pump protein, N. pharaonis halorhodopsin (NpHR), into an ND using the native archaeal lipids or artificial lipids to investigate its photochemical properties under the physiological conditions. Although various kinds of HRs have been discovered and reported (6), NpHR is advantageous to use in this research. When compared to the other microbial rhodopsins described above, NpHR is advantageous because 1) a large amount of NpHR can be obtained from the native archaeon N. pharaonis KM-1 strain, which was generated by ultraviolet (UV) mutagenesis from the N. pharaonis DSM2160^T strain to overproduce NpHR (allowing for the contamination of intrinsic NpSRII to be ignored (16)); 2) NpHR forms a robust trimer that is stable in the presence of detergents such as n-dodecyl-β-D-maltoside (DDM) and n-octyl-β-D-glucoside (17); and 3) as we have already reported, the spectroscopic properties of native NpHR in KM-1 membrane fragments were examined extensively (18).

Sasaki and co-workers confirmed that NpHR purified from N. pharaonis KM-1 forms a trimer after the DDM treatment (19). Preparing ND-reconstituted proteins requires solubilization by detergents, as illustrated in Fig. 1. Unlike NpHR, trimeric HsBR dissociates into monomers in the presence of detergents (20). GPR oligomeric assembly is also altered by detergents (21). In native membranes, NpHR is densely distributed on the cytoplasmic membrane and structural changes of a trimer undergoing photoreaction alters both the structure and photoreaction activity of adjacent trimers (22). Comparison of the photoreaction activity between ND-reconstituted NpHR and NpHR in native membrane fragments will allow us to investigate the functional significance of trimer-trimer interactions. The following study has obtained multiple new, to our knowledge, structural and functional properties of NpHRs that will aid our understanding of the molecular mechanisms of rhodopsin photoreaction cycles.

Figure 1.

Illustrations for the reconstitution of NpHR into (a) NL-NDs and (b) POPC-NDs. Details for ND construction are described in Materials and Methods. To see this figure in color, go online.

Materials and Methods

Protein expression and purification

Native NpHR in membrane fragments (MF NpHR) was obtained from a halo-alkaliphilic archaeon N. pharaonis KM-1 strain (a kind gift from Dr. Kunio Ihara), which overexpresses NpHR (23). The procedures for culture and purification of the native membrane fraction were the same as those reported in our previous study (18).

For preparation of recombinant NpHR, the plasmid construction, protein expression procedure, and purification protocols were performed as reported previously (24,25). Recombinant NpHR has the additional Val-Asp-His₆ sequence in the C-terminus because of the His-tag-containing pET21c vector (Novagen, Madison, WI). Recombinant NpHR was solubilized in 10 mM MOPS (pH 7.0) containing 0.1% DDM (Dojindo Laboratories, Kumamoto, Japan) and 0.1 mM–1.0 M NaCl. The expression and purification of MSP1D1 were performed as previously described (26). Purified MSP1D1 also has the additional Gly-Pro-His₆ sequence in the N-terminus.

Reconstitution of native NpHR into an ND using native archaeal lipids

To prepare ND using the native archaeal lipids (NLs), the native membrane from N. pharaonis KM-1 was washed three times with the ND buffer (20 mM Tris-HCl (pH 7.4), 100 mM NaCl) by ultracentrifuge (4°C, 50,000 rpm, 3 h). Afterwards, the membrane containing native NpHR was suspended in ND buffer containing 1% DDM with MSP1D1.

We used MSP1D1 as the scaffold protein to form NDs. MSP1D1 was shown to form an ND with a bilayer area of 44 nm² (2,27), which is suitable for trimeric NpHR, the transmembrane area of which is 24 nm² (28). After the addition of MSP1D1, the solution containing the native MFs, including native NpHR and MSP1D1, was gently rotated at 4°C for 2 h. Then, sufficient amounts of BioBeads SM2 (Bio-Rad, Hercules, CA) previously equilibrated with ND buffer were added to 1 mL of the MF to remove DDM. After gentle rotation of this suspension at 4°C for 12 h, the beads were removed by sedimentation. The supernatant was purified by size-exclusion chromatography using a Superdex 200 10/300 GL column (GE Healthcare, Chicago, IL) previously equilibrated with the ND buffer. The eluted protein was monitored at 280 and 580 nm.

The optimal ratio of NpHR/MSP1D1 was determined by the chromatogram from the size-exclusion column, which depends on the yield of ND-reconstituted NpHR. For the estimation of the molecular weight by the size-exclusion chromatogram, we created a calibration curve according to the manufacture’s protocol. After purification by the size-exclusion chromatography, the ND buffer was exchanged with 10 mM MOPS (pH 7.0) by ultrafiltration using an Amicon Ultra filter (30,000 MWCO; Millipore, Burlington, MA). The concentration of NpHR was adjusted to 10 μM by measuring its absorption maximum at 578 nm. When necessary, NaCl was added to achieve a final concentration of 0.1 mM–1.0 M. The extinction coefficient of 54,000 M⁻¹ cm⁻¹ at 578 nm (29) was used to estimate the concentration of NpHR in the presence of Cl⁻.

Reconstitution of recombinant NpHR into a ND using 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

To reconstitute NpHR into a ND, we used 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) as the lipid inside the ND. As previously reported (30), archaeal lipids are generally composed of a core lipid (archaeol or caldarchaeol) and phosphodiester-bonded polar headgroups or glycosides that are linked to one of the core lipids. Although the lipids having phosphodiester-bonded polar headgroups are negatively charged, the glycoside-linked lipids are neutral, resulting in the partially, not entirely, negatively charged membrane surface. NDs using artificial lipids linked to negatively charged headgroup such as 1-palmitoyl-2-oleyl-sn-glycero-3-phospho-1-glycerol would be a better model of the archaeal membrane, but the high-anionic-content NDs exhibit polyelectrolyte behavior, showing irreversible aggregation in the presence of metal cations (31). On the other hand, NDs using lipids linked to zwitterionic headgroups such as POPC or POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine) were unaffected by high concentrations of metal cations. Stable NDs using POPC or other lipids linked to zwitterionic headgroups have been utilized for the reconstitution of rhodopsin and its analog proteins to characterize their spectroscopic properties (12,32).

For the preparation of an ND containing POPC, POPC (Avanti Polar Lipids, Alabaster, AL) was dissolved in chloroform at a concentration of 100 mM. 100 μL of the POPC/chloroform solution was dried in a vacuum-centrifugal evaporator for ∼12 h. A thin film of the dried lipid was solubilized in ND buffer with 1% DDM to adjust the POPC concentration to 50 mM. The following procedures were repeated three times to obtain a clear and solubilized POPC solution: 1) incubation at 37°C for 5 min, 2) vortexing for 5 min, 3) sonication for 5 min, and 4) freezing at −80°C for 10 min.

We used recombinant NpHR expressed in E. coli to prepare the POPC-NDs. The procedures for the expression and purification of recombinant NpHR were as previously reported (24,25). We also prepared DDM-solubilized recombinant NpHR (DDM NpHR) as previously reported (17). To reconstitute recombinant NpHR into an ND, recombinant NpHR and MSP1D1 were solubilized in the ND buffer containing 1% DDM. The procedures for the further purification of the POPC-ND-reconstituted recombinant NpHR (POPC-ND NpHR) were the same as those used for NL-ND NpHR.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using standard methods. Proteins were detected using the Coomassie brilliant blue staining method. 15% acrylamide gel (e-PAGEL, E-T15L) and molecular markers (XL-Ladder Broad Range) were purchased from ATTO, Tokyo, Japan, and APRO Life Science Institute, Tokushima, Japan, respectively.

Spectroscopic measurements

All the spectroscopic measurements in this study were carried out at 25°C. The concentrations of all NpHR samples were adjusted to 10 μM. The buffer used for the measurements consisted of 10 mM MOPS (pH 7.0), 0 or 0.1% DDM, and 0.100 mM–4.00 M NaCl.

UV-Vis absorption spectra were measured by using a UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). Samples were put into a 10 mm optical quartz cuvette, and the baseline was corrected using a background spectrum of the buffer solution without protein. The Cl⁻ titration experiments for the initial state were carried out by varying the concentrations of NaCl from 0.100 mM to 1.00 M (17,18). The absorption difference from NpHR without Cl⁻ at 633 nm (ΔA) were plotted against the Cl⁻ concentrations ([Cl⁻]) and fitted by the following equation:

$$\Delta A=\Delta A_{\text{max}}\times \frac{{\left[{\text{Cl}}^{-}\right]}^n}{K_{\text{d},\text{initial}}^n+{\left[{\text{Cl}}^{-}\right]}^n}$$

where ΔA_max is the maximal absorbance change for the Cl⁻ binding, K_d,initial is the dissociation constant for Cl⁻ in the initial state, and n is the Hill coefficient.

CD spectra were measured using a Jasco J-725 spectropolarimeter (Jasco, Tokyo, Japan) in the 300–700 nm regions at a scanning speed of 200 nm/min. For each measurement, the accumulation was conducted four times.

Flash photolysis measurements were carried out using a computer-controlled laser flash photolysis apparatus equipped with an Nd:YAG laser (532 nm, 7 ns) as described in previous reports (17,18). The data collected before laser pulsing were adopted as the baseline. 30 accumulations were obtained for each sample. The Cl⁻ titration experiments to measure the photointermediate states were carried out by varying the concentrations of NaCl from 0.0200 to 4.00 M.

Analysis of flash photolysis data

Global fitting analysis was performed for the data sets measured from 410 to 710 nm in 10 nm intervals. The details of the procedure were reported previously (17). Briefly, the data were fitted using a multiexponential function simultaneously for the entire data set. The appropriate number of exponents was determined to be four, based on the reductions in the standard deviation of the residuals (33). Further analysis proceeded according to the following sequential model (18,33):

$${\mathrm{P}}_0\to {\mathrm{P}}_1\to {\mathrm{P}}_2\to {\mathrm{P}}_3\to {\mathrm{P}}_4\to {\mathrm{P}}_0$$

where P₀ represents the initial state and P₁, P₂, P₃, and P₄ represent the four kinetically defined intermediate states (18,33). This model contains only the forward reactions from the P₁ to P₄ states. Based on visible absorption and electrogenicity kinetic studies, Váró et al. and Ludmann et al. proposed the following scheme for the NpHR photocycle consisting of six photointermediate states (34,35):

$$\mathrm{N}\mathrm{p}\mathrm{H}\mathrm{R}\to \mathrm{K}\leftrightarrow {\mathrm{L}}_1\leftrightarrow {\mathrm{L}}_2\leftrightarrow \mathrm{N}\leftrightarrow \mathrm{O}\leftrightarrow \mathrm{N}\mathrm{p}\mathrm{H}{\mathrm{R}}^{\text{'}}\to \mathrm{N}\mathrm{p}\mathrm{H}\mathrm{R}$$

Thus, these P₁–P₄ states may contain a few physically defined photointermediates of the photocycle, such as L₁, L₂, N, and O, when the reverse reactions exist. Using the fitting results, the time constant τ₁–τ₄ and the absorption differences of the P₁–P₄ states from P₀, (Δε₁–Δε₄), were determined. The P₀ spectrum was obtained by subtracting the background scattering from the spectrum of the initial state (18). Finally, the absolute spectra of the P₁–P₄ states were obtained by adding the spectrum of P₀ to the absorption differences, Δε₁–Δε₄. For details, see the work by Chizhov et al. (33) as well as our previous studies (17,18). The mathematical description was given by Sato et al. (25).

Decomposition of the P₃ spectra and determination of K_d,N↔O

During the photoreaction cycle of NpHR, the P₃ state is a quasiequilibrium mixture of N and O intermediates whose relative fractions dependent on the Cl⁻ concentration (18):

$$\text{N}\leftrightarrow \text{O}+{\text{Cl}}^{-}$$

where the dissociation constant of chloride, K_d,N↔O, is described as

$$K_{\text{d},\text{N}\leftrightarrow \text{O}}=\frac{\left[\text{O}\right]\left[{\text{Cl}}^{-}\right]}{\left[\text{N}\right]}$$

In addition to the main absorption bands, the P₃ spectrum includes the β-bands from N and O in the visible region. Assuming both β-bands are identical with that of the P₀ state, the P₃ spectrum is expressed as

$${\text{Spectrum of P}}_3=f\times Abs\left(\text{N},\lambda \right)+\left(1-f\right)\times Abs\left(\text{O},\lambda \right)+Abs\left(\beta ,\lambda \right)$$

where f, Abs(N, λ), Abs(O, λ), and Abs(β, λ) represent the absorbances for the fractions of N in the P₃ state, the main band of N, the main band of O, and the β-band, respectively (17,18). By employing skewed Gaussian functions to express the three absorption bands, we deconstructed the P₃ spectra to estimate f. In this calculation, we assumed f to be the only parameter varying with the Cl⁻ concentration (18).

Values of f and 1 − f (fractions of N and O in the P₃ state, respectively) were plotted against the Cl⁻ concentration to determine the Cl⁻ binding affinity. In these plots, the two curves crossed at f = 0.5 (the plots for 1 − f were not shown). The concentration of Cl⁻ giving f = 0.5 corresponds to K_d,N↔O.

Results

Reconstitution of NpHR trimer into a ND using native lipids or POPC

Although N. pharaonis has two kinds of rhodopsins, NpHR and NpSRII, the mutant strain KM-1 preferentially overexpresses NpHR in the cytoplasmic membrane, together with a red-colored C₅₀ carotenoid, bacterioruberin (Brub). In the presence of Cl⁻, NpHR is in its purple form; absorbing 578 nm (orange) light makes the membrane fraction appear red-purple (18). To optimize the conditions for NL-ND reconstitution (Fig. 1 a), we prepared six samples containing NpHR and MSP1D1 in molar ratios of 1:1, 2, 5, 10, 15, and 20 as previously reported for HsBR (11). After the addition of 1% (w/v) DDM and BioBeads, the formation of ND-reconstituted NpHR was confirmed using size-exclusion chromatography and monitored by its absorbance at 280 and 580 nm. In the cases of NpHR/MSP1D1 = 1:1 and 1:2, only one peak in the column void volume was observed, indicating the aggregation of these proteins. Increasing the ratio of NpHR/MSP1D1 (NpHR/MSP1D1 = 1:5–20) elicited a peak around the 12 mL elution volume that appeared at both 280 and 580 nm, as shown in Fig. 2 a (indicated by an arrow). This fraction was clear and red-purple colored in the absence of DDM, indicating the coexistence of NpHR and Brub in the NL-ND. Because we successfully obtained sufficient NL-ND sample in our preparation of NpHR/MSP1D1 = 1:10 (Fig. 2 a), we used this ratio for further preparations of NL-ND NpHR.

Figure 2.

Preparation of reconstituted NpHR into NL-NDs and POPC-NDs. (a and c) Size-exclusion chromatograms of (a) NL-NDs (native N. pharaonis KM-1 membrane/MSP1D1 = 1:10) and (c) POPC-NDs (recombinant NpHR/MSP1D1/POPC = 1:2:60) are given. Single and double asterisks indicate the void volume of the size-exclusion column and empty NDs, respectively. Arrows indicate NpHR embedded in NDs. The fractions around the 12 mL elution volume were used for experiments. The experiments were carried out using 20 mM Tris-HCl (pH 7.4) buffer containing 0.1 M NaCl. (b and d) Absorption spectra of (b) NL-NDs and (d) POPC-NDs for reconstituted NpHR from the fractions indicated by arrows in (a) and (c), respectively, in buffer containing 10 mM MOPS (pH 7.0) and 1.0 M NaCl are shown. The absorption spectra of DDM NpHR are depicted in the gray broken lines. In (b), absorption spectrum of Brub (red) and calculated spectrum of NpHR (blue) from the observed spectrum (black) are also displayed. (e) SDS-PAGE of molecular markers (lane 1), native NpHR (lane 2), recombinant NpHR (lane 3), native NpHR in NL-ND (lane 4), recombinant NpHR in POPC-ND (lane 5), and empty POPC-ND (lane 6) is shown. To see this figure in color, go online.

To investigate the environmental structure of the chromophore binding site in NL-ND NpHR, we again measured the absorption spectra. Fig. 2 b shows the absorption spectrum for the 12 mL elution fraction (black). The subtraction of the Brub absorption spectrum (red) from the observed spectrum (black) resulted in a similar spectrum (blue) to that of DDM NpHR (dotted), demonstrating that NpHR was reconstituted into the NL-ND with Brub. The absorption peaks of NL-ND NpHR appeared at 578 and 600 nm in the presence of 1.0 M Cl⁻ and the absence of Cl⁻, respectively. A similar shift of the absorption peak by the addition of Cl⁻ was also observed in DDM NpHR measurements (Table 1; (18)).

Table 1.

Absorption Maxima in the Visible Region and Affinity to Chloride of NpHR

	λmax [nm]		Kd [mM]
	0 M Cl−	1.0 M Cl−	Cl− Binding (Kd initial)	Cl− Release (Kd,N↔O)
DDM	600	578	2.9a	1200
POPC-ND	600	578	3.5	269
NL-ND	600	578	16	290
MF	600	578	6.1a	3700a

λ_max, absorption maxima. K_d, dissociation constant.

^aFrom Kikukawa et al. (18).

We also prepared POPC-ND NpHR to evaluate the contribution of lipids to the photoreaction of NpHR when compared with NL-ND NpHR. In this preparation, we used recombinant NpHR purified in the presence of DDM (Fig. 1 b). By following the procedure for reconstituting HsBR trimer (11), we prepared seven samples containing recombinant NpHR, MSP1D1, and POPC in the molar ratios of 1:2:10, 20, 40, 60, 80, 100, or 120, respectively. For NpHR/MSP1D1/POPC = 1:2:40 and 60, a single peak at the 12 mL elution volume was observed, as found for NL-ND NpHR (Fig. 2 c). The absorption spectrum of this fraction is displayed in Fig. 2 d, demonstrating that the absorption intensity at 578 nm was not changed but that at 280 nm was increased when compared to DDM NpHR. This change is due to the presence of MSP1D1, a scaffold protein for NDs, in POPC-ND NpHR. We also confirmed that the addition of 1.0 M Cl⁻ shifted the peak from 600 to 578 nm in the absorption spectrum of POPC-ND NpHR. These results imply that the structure around retinal chromophore and its structural changes upon Cl⁻ binding in POPC-ND NpHR was also similar to those in DDM NpHR.

The reconstitution of NpHR into NDs was also supported by SDS-PAGE gels stained with Coomassie blue (Fig. 2 e). The bands of native NpHR (lane 2) and MSP1D1 (lane 6) were overlapped because of the similar mobility (lane 4), although the molecular weight of native NpHR is smaller than that of MSP1D1. Recombinant NpHR (lane 3), which has an additional eight amino acid residues at the C-terminal, exhibits slower mobility than that of MSP1D1 as expected, resulting in two bands (lane 5).

Oligomeric assembly of NpHR in NDs

As previously reported (17,36), NpHR forms a trimeric assembly under the physiological conditions. To confirm that trimeric NpHR was reconstituted into the ND, we measured their visible CD spectra. Visible CD spectroscopy is a well-established method for evaluating the oligomeric state of microbial rhodopsins such as NpHR (25,36,37), H. halobium BR (38,39), Gloeobacter rhodopsin (40), Anabaena SR (41), and thermophilic rhodopsin (42). Time-dependent density functional theory calculation on the retinal protonated Schiff base chromophore revealed that the visible CD spectrum of BR is dominated by the exciton interactions based on the distance between two retinals in the oligomeric states of BR (38), showing that positive and negative CD bands at 530 and 570 nm, respectively, are characteristic of the tetrameric states of BR.

In DDM solution (Fig. 3 a), the CD spectrum contains biphasic bands at 540 nm (positive) and 600 nm (negative) with a crossover point at 580 nm, indicating that DDM NpHR forms a trimer (17). Given their similar CD spectra (Fig. 3 b), trimer formation was also observed for POPC-ND NpHR. Different CD spectra were obtained for NL-ND NpHR (Fig. 3 c) and MF NpHR (Fig. 3 d), particularly the two CD bands that are emitted from the carotenoid Brub in the 450–550 nm region (18). In addition to these bands, a small negative CD band at 600 nm was observed, which corresponds to the trough around 600 nm in the CD spectra of DDM NpHR solution, confirming the trimeric structure of NL-ND and MF NpHR. The positive CD band around 540 nm in recombinant NpHR is also overlapped with the CD bands from Brub in NL-ND NpHR. In addition, the molar ellipticity of Brub for NL-ND NpHR was significantly small when compared to MF NpHR, suggesting that some of the Brub in the native membranes washed out during the purification step for NL-ND NpHR.

Figure 3.

Visible CD spectra of NpHR in (a) DDM, (b) POPC-NDs, (c) NL-NDs, and (d) native N. pharaonis KM-1 membrane (MF NpHR). The samples were suspended in 10 mM MOPS (pH 7.0) buffer containing 0 or 0.1% DDM and 1.0 M NaCl.

Cl⁻ binding in the initial state

The Cl⁻ binding processes for ND-reconstituted NpHRs were examined by absorption spectra under various Cl⁻ concentrations as displayed in Fig. 4. The spectral changes in the Cl⁻ titration for NL-ND NpHR (Fig. 4, a–c) and POPC-ND NpHR (Fig. 4, d–f) are illustrated. As the Cl⁻ concentration increases, the absorption maximum was shifted from 600 to 578 nm in both cases, although the peaks in the presence of Cl⁻ were not clear because of the overlapping absorption of Brub in NL-ND NpHR (Fig. 4, a and d). A similar peak shift in the absorption spectrum was previously reported for DDM NpHR (17) and MF NpHR (18). In the difference spectra for NL-ND NpHR (Fig. 4 b) and POPC-ND NpHR (Fig. 4 d), an isosbestic point was clearly observed at 590 nm, indicating a single-step transition of NpHR from the Cl⁻-unbound to Cl⁻-bound states. For NL-ND NpHR (Fig. 4 b), the Cl⁻-dependent absorption change was also detected in the 400–550 nm range, which can be assigned to the absorption change of Brub under these conditions (18). The amplitudes for the difference absorbance at 633 nm were plotted against the Cl⁻ concentration (Fig. 4, c and f) to determine the dissociation constants of the Cl⁻ binding in the initial state, K_d,initial, using Hill’s equation as described in the Materials and Methods. As depicted in Fig. 4, c and f, the K_d,initial-values were determined to be 16 and 3.5 mM for NL-ND NpHR and POPC-ND NpHR respectively. These values were quite similar to MF NpHR (6.1 mM) (18) and DDM NpHR (2.9 mM) (18). The K_d,initial-values for Cl⁻ binding are summarized in Table 1.

Figure 4.

Chloride ion titration experiments for NpHR in NL-NDs (a–c) and POPC-NDs (d–f). The samples were suspended in 10 mM MOPS (pH 7.0) buffer containing 0.2–2000 mM NaCl. (a and d) Chloride-ion-dependent blueshift (indicated by arrows) in the absorption spectra is shown. (b and e) Chloride-ion-dependent spectral changes in the difference absorption spectra are shown. (c and f) The difference absorbance at 633 nm is plotted against the Cl⁻ concentration logarithmically. The titration curves were analyzed using Hill’s equation. (e) K_d,initial for NL-ND NpHR was calculated to be 16 mM with a 0.77 Hill coefficient. (f) K_d,initial for POPC-ND NpHR was calculated to be 3.5 mM with a 0.81 Hill coefficient. To see this figure in color, go online.

Photoreaction kinetics

After the Cl⁻ binding, the irradiation of light activates the photoreaction cycle in NpHR. To monitor the photoreaction cycle of ND-reconstituted NpHRs, we performed flash photolysis measurements. Fig. 5 depicts the flash-induced light-minus-dark difference spectra (Fig. 5, a and b) over the time course of the absorption changes at selected wavelengths (Fig. 5, c and d) for NL-ND NpHR (left) and POPC-ND NpHR (right) in the presence of 1.0 M NaCl. The time for NL-ND NpHR activity (Fig. 5 a; 10 μs–1.4 s) was significantly longer than POPC-ND NpHR (Fig. 5 b; 10 μs–204 ms, Table 2). After the flashlight excitation, the intensity of absorption at 580 nm decreased when compared to the initial state, whereas the intensity of the absorption at 500 nm from L (L₁ and L₂) was enhanced. Note that M-intermediate (M), which appears after L in proton-pumping rhodopsins including BR, is lacking for NpHR. Over time, the absorption band for L decreased with a concomitant increase in the 650 nm absorption band for O, which we assign to the Cl⁻-released form of NpHR photointermediate states. Previous studies reported that N, which still binds Cl⁻ in the cytoplasmic binding site, appears at 500 nm after the disappearance of L and is in the equilibrium with O in a Cl⁻-concentration-dependent manner (18). Finally, the intensity of the absorption band from the initial state at 580 nm was restored with a concomitant decrease in that of O as the photocycle finished (18,34,35).

Figure 5.

Photoreaction of NpHR in (a and c) NL-NDs and (b and d) POPC-NDs. The samples were suspended in 10 mM MOPS (pH 7.0) buffer containing 1.0 M NaCl. (a and b) Flash-induced difference spectra over time are shown. The arrows indicate the direction of the time-dependent absorption changes. (c and d) The time course of the absorption changes at selected wavelengths. The photointermediates L and O and the original pigment (retinal) were monitored at 500 nm (red), 650 nm (blue), and 580 nm (green). The simulation curves consisting of the sum of four exponents are also shown in black lines. To see this figure in color, go online.

Table 2.

Time Constants of Photoreaction Kinetics in the Presence of 1.0 M NaCl and 0.1 M NaCl Shown in Brackets

	NL-NDs	MFs	DDM	POPC-NDs
τ1	0.37 (0.29)	0.32 (0.31)	0.28 (0.26)	0.24 (0.21)
τ2	1.1 (2.0)	1.1 (1.1)	0.44 (0.56)	0.31 (0.66)
τ3	4.2 (8.0)	4.5 (44)	0.83 (3.1)	1.1 (1.3)
τ4	27 (41)	47 (237)	39 (24)	59 (17)

Time constants expressed in milliseconds.

Our recent study revealed that the flash-induced absorption change of Brub is also observed during the transition from N to O in MF NpHR (18). Because Brub is sensitive to local environmental structure changes, the absorption change reflects the altered electrostatic environment and/or distortion of its own configuration during the Cl⁻-release process (i.e., from the cytoplasmic binding site to the bulk cytoplasmic space). However, the characteristic absorption change of Brub was not observed in NL-ND NpHR (Fig. 5 a). This is probably due to the removal of Brub located near the Cl⁻-releasing site during the purification for NL-ND NpHR, as suggested by suppressed spectral changes in the visible CD spectrum for NL-ND NpHR (Fig. 3 c) when compared with MF NpHR (Fig. 3 d), and/or different NpHR-Brub interactions in NL-ND NpHR.

To gain further insight into photocycle intermediates in ND-reconstituted NpHRs, we performed a global fitting analysis on our flash photolysis measurements. For fitting, we used the exponential decay function with a sum of four exponential terms as described in Materials and Methods (17). Based on the sequential model (18), we calculated the absorption spectra of these kinetically defined states, designated P₁–P₄ (Fig. 6). As described in the Materials and Methods, P₁–P₄ contain kinetically defined photointermediates such as L, N, and O. As reported in our previous studies (18), we assigned four calculated spectra to the photointermediates in the kinetically defined P₁–P₄ states, as indicated in Fig. 6. In this figure, P₀ denotes the absorption spectrum of the initial state of NpHR. P₁ and P₂ have an absorption maximum at 500 nm corresponding to L₁ and L₂, respectively. P₃ contains two peaks at 500 and 650 nm, which can be assigned to N and O, respectively. P₄ absorbs at 580 nm similar to the initial state P₀, which can be assigned to NpHR completion of the photocycle (18).

Figure 6.

The calculated absorption spectra for the kinetically defined P_i states (P₁–P₄) for NpHR incorporated into (a) NL-NDs and (b) POPC-NDs. P₀ denotes the absorption spectrum of original unphotolyzed NpHR. Gray broken lines denote the P₃ spectra for DDM NpHR. To see this figure in color, go online.

Although the same number of exponents can be used for the kinetic analysis of all NpHR systems in this study, the time constants were dependent on the system, as summarized in Table 2. As listed in Table 2, the photoreaction kinetics of NL-ND NpHR is similar to that of MF NpHR but significantly different from those of DDM and POPC-ND NpHR. In the presence of 1.0 M Cl⁻, the τ₃ transition from the P₃ to P₄ states is quite different: τ₃ of NL-ND and MF NpHR is ∼4 ms, whereas DDM NpHR and POPC NpHR are much shorter (∼1 ms). Thus, the transition from the P₃ to P₄ states is significantly affected by membrane environments surrounding NpHR and is slower under native membrane conditions. Furthermore, under diluted Cl⁻ concentration conditions (0.1 M), the time constants for the transitions from the P₃ to P₄ states and from the P₄ to P₀ states (τ₃ and τ₄, respectively) for NL-ND NpHR were much shorter than those in MF NpHR. The photocycle of NL-ND NpHR was accelerated in the presence of 0.1 M Cl⁻ when compared to MF NpHR.

Cl⁻ releasing in photointermediate states

We next focused on the Cl⁻ dependency of the P₃ spectra to examine the Cl⁻-releasing process. As described above, two photointermediates, N and O, are in a Cl⁻-concentration-dependent quasiequilibrium (i.e., the Cl⁻-bound and Cl⁻-released forms (43,44)). Cl⁻ is released from the cytoplasmic binding site to the cytoplasmic bulk space during the photoreaction process from N to O. Thus, the dissociation constant for the Cl⁻ release (K_d,N↔O) can be estimated from the Cl⁻ concentration dependence of the P₃ spectra (18). Fig. 7 displays the fraction of N in the P₃ state sigmoidally increased as Cl⁻ concentration increases. Cl⁻ concentration dependence of the N fraction was analyzed using Hill’s equation, as described in the Materials and Methods. K_d,N↔O, corresponding to the quasiequilibrium constants between N and O for DDM, POPC-ND, NL-ND, and MF NpHRs are summarized in Table 1. The K_d,N↔O-values for NL-ND and POPC-ND NpHRs were 0.290 and 0.269 M, respectively, which were ∼4 and 12 times smaller than those of DDM (K_d,N↔O = 1.2 [M]) (17) and MF NpHRs (K_d,N↔O = 3.7 [M]) (18). These results strongly suggest that the affinity of the cytoplasmic Cl⁻-binding site of NpHR is increased by reconstitution to NDs.

Figure 7.

The fraction of N-intermediate calculated from the P₃ Cl⁻-dependence spectra. The data were analyzed using Hill’s equation as described in the Materials and Methods. To see this figure in color, go online.

Discussion

Reconstitution of a single NpHR trimer into an ND using NLs

To confirm that a single trimeric NpHR was reconstituted into an ND, the apparent molecular weight of NpHR was estimated from the elution profile from size-exclusion chromatography, as shown in Fig. 2. The elution peak at 12 mL in Fig. 2 c indicates that the apparent molecular weight of POPC-ND NpHR is ∼210 kDa. Considering that the molecular weight of MSP1D1 is ∼44 kDa, the apparent molecular weight of recombinant NpHR and POPC is ∼170 kDa. We used MSP1D1 to form NDs with inside and outside diameters of ∼8 and 10 nm with a bilayer area of ∼50 nm² (45). Assuming that a single trimeric NpHR whose transmembrane area is ∼20 nm² is reconstituted into NDs and the mean surface area of POPC is 0.7 nm², the number of POPC would be ∼90 (= (50 − 20 nm²)/0.7 nm² × 2) and the total molecular weight of POPC (= 760) incorporated into NDs would be ∼70 kDa. Therefore, the apparent molecular weight of recombinant NpHR is ∼100 kDa, corresponding that of trimeric recombinant NpHR (97.2 kDa). Thus, it appears that a single trimeric NpHR is reconstituted into both POPC-ND and NL-ND. As discussed above, the negative CD band around 600 nm is characteristic of the trimeric states of the rhodopsin family, confirming a trimeric assembly of NpHR in NL-NDs and POPC-NDs (Fig. 3), although one of the characteristic CD bands (∼540 nm) of the trimeric state was not detected because of the overlapping of the intense positive CD bands from Brub in the CD spectrum for NL-ND NpHR, and we cannot exclude the possibility of significant conformational differences between trimeric NpHR in NL-NDs and that in POPC-NDs. Taken together, the results from size-exclusion chromatography and CD spectra clearly indicate that a single trimeric NpHR was successfully reconstituted into both NL-NDs and POPC-NDs using MSP1D1.

Cl⁻-binding and releasing processes in ND-reconstituted NpHR

As previously reported (46), Cl⁻ binding to NpHR induces structural changes around the retinal chromophore that are characterized by the absorption peak shift from 578 to 600 nm in DDM NpHR and MF NpHR. The reconstitution of NpHR into NL-ND or POPC-ND did not affect this peak shift (Fig. 4), indicating that the interactions between the protein surface of NpHR and lipids or detergent did not influence the structural changes of the retinal chromophore upon the Cl⁻ binding. The shift of the absorption peak from 578 to 600 nm by the addition of Cl⁻ also supports the formation of trimeric NpHR in NL-ND or POPC-ND because monomeric NpHR exhibits absorption peaks at 578 and 574 nm in the presence and absence of Cl⁻, respectively (17).

The dissociation constant for Cl⁻ binding in the initial state, K_d,initial, is one of primary indicators for assaying the pumping function of NpHR. As listed in Table 1, larger K_d,initial was observed for NpHR embedded in NLs—NL-ND NpHR (16 mM) and MF NpHR (6.1 mM)—compared with those in the other systems—POPC-ND NpHR (3.5 mM) and DDM NpHR (2.9 mM). Although we have not yet fully understood the structural factors that affect the K_d,initial-values, negative charges on the membrane surface could be one of the factors to reduce the binding affinity of Cl⁻ in NL-embedded NpHR because of the repulsive electrostatic interactions between the negatively charged headgroup of the NL and the anionic Cl⁻ ion. A previous study also reported that the imposition of the negative membrane potential suppressed Cl⁻ binding to the extracellular surface in NpHR embedded in the archaeal membrane (18). The photoreaction kinetics in HsBR also depends on the charge on the membrane surface (14). In NL-ND NpHR, some of the neutral Brub was lost during the reconstitution into NDs, resulting in more enhanced negative charge on the membrane surface and more reduced Cl⁻ binding affinity (K_d,initial = 16 mM). Thus, the interactions between NpHR and the surrounding molecules might affect the structure or structural changes associated with the initial Cl⁻ binding at the extracellular side, but such effects would not be so drastic.

In sharp contrast to K_d,initial, the quasiequilibrium constant between N and O, K_d,N↔O, was more substantially dependent on interactions between NpHR and the surrounding molecules. The K_d,N↔O-values for NL-ND and POPC-ND NpHRs (0.290 and 0.269 M) were much smaller than those of DDM (1.2 M) (17) and MF NpHRs (3.7 M) (18). One possible reason for smaller K_d,N↔O of ND-reconstituted NpHRs would be the limitation of the structural changes associated with light illumination of NpHR in NDs. As evident by the x-ray structure using a C2 crystal of NpHR that was soaked in a postcrystallization solution containing bromide ions (47), upon the formation of N with a bromide ion bound to the cytoplasmic vicinity of the retinal Schiff base, the cytoplasmic half of helix F was found to move outward to create a water channel in the cytoplasmic interhelical space, whereas the extracellular half of helix C moves inward. In atomic force microscopy (AFM) images of the cytoplasmic surface of mutant HsBR during a slow photoreaction cycle, a prominent protrusion from the interhelical loop on the cytoplasmic surface was also observed (22). Comparison of the AFM images obtained before and during light illumination revealed that the protruding areas around helices E and F shift outwards from the trimer center under illumination. Such conformational changes, including such outward displacements, would be suppressed in NDs because of the surrounding lipids being wrapped by the ND scaffold protein (MSP1D1). Thus, the flexibility of the lipid bilayer in the native membrane would facilitate conformational changes during the Cl⁻-releasing process, which reduces the Cl⁻ binding affinity of NpHR, promoting Cl⁻ release to the cytoplasmic site.

The trimer-trimer interactions in native NpHR-containing MFs under physiological conditions could be another factor that could reduce K_d,N↔O of ND-reconstituted NpHRs. Although detailed distribution of native NpHR on the cytoplasmic surface under physiological conditions has been unclear, NpHR is thought to be densely expressed in the native membrane as observed for HsBR (22), which is supported by the crystallographic structure of NpHR showing lateral contacts between neighboring trimers (28,48). The theoretical calculation suggested that intertrimeric exciton couplings induced by the retinals in neighboring trimeric NpHRs reduced the overall intensity of the exciton couplet by a factor of ∼2, resulting in significant changes of the intensity of the CD bands in the visible region (38). The functional significance of the trimer-trimer interactions in NpHR is also not clear, but the lack of trimer-trimer interactions in HsBR trimers embedded in the purple membrane of H. salinarum significantly reduced the rate of the conformational changes under continuous light illumination. In disassembled mutant HsBR, formation of the outwardly displaced E-F loop intermediate was suppressed, and the photoexcited state was readily de-excited to the ground state without outward displacement (49). The photoreaction cycle of NpHR is not exactly the same as that of HsBR because of the lack of the M-intermediate state. However, the de-excitation of the photoexcited state of NpHR before outwards displacement suggests that regeneration of the ground state is occurring without releasing Cl⁻ corresponding to the apparent high affinity of Cl⁻ binding, thus reducing Cl⁻ transport efficiency. We propose that the lack of trimer-trimer interactions reduces the K_d,N↔O-values for NL-ND and POPC-ND NpHR. In other words, the trimer-trimer interactions in NpHR embedded in the flexible native membrane would enhance the Cl⁻-releasing process by allowing outwards displacement of NpHR helices, which facilitates efficient Cl⁻ transport.

Dependence of photochemical properties on surrounding environments of NpHR

As discussed above, the reduced K_d,N↔O-values for NL-ND and POPC-ND NpHR imply that the reconstitution of NDs of NpHR perturbed the photocycle of NpHR. To gain further insights into the perturbation of the photocycle in ND-reconstituted NpHR, we focused on the lifetimes of the intermediates in the photoreaction cycle. Table 2 summarizes the time constants for the decay of intermediates in the NpHR photocycle. As shown in Table 2, the lifetimes of the intermediates highly depend on the surrounding environments of NpHR, and the time constants of NpHR can be categorized into two groups: NL-ND NpHR/MF NpHR and POPC-ND NpHR/DDM NpHR. In the presence of 1.0 M Cl⁻, the two groups show similar time constants τ₁ and τ₄, which correspond to the transitions from the P₁ to P₂ states and the P₄ to P₀ states, respectively. Conversely, substantial differences were observed for the time constants τ₂ and τ₃ and the transitions from the P₂ to P₃ states and from the P₃ to P₄ states. It should be noted here that the transitions from the P₂ to P₃ states and from the P₃ to P₄ states corresponds to the transitions from L₂ to N and from O to NpHR′, both of which would be required for conformational changes in NpHR (47). During the transition from L₂ to N, Cl⁻ is transported from the extracellular site to the cytoplasmic site in NpHR. The Cl⁻ releasing from NpHR to the cytoplasm and the Cl⁻ rebinding from the extracellular space to NpHR proceed in the transition from O to NpHR′. As previously reported (30) and discussed in the previous section, the archaeal membrane surface is negatively charged, and repulsive electrostatic interactions are formed with Cl⁻, which would suppress the Cl⁻ uptake rate from the extracellular space to NpHR, leading to longer τ₃ in NL-ND (1.1 ms) and MF NpHR (1.1 ms) compared with those of POPC-ND (0.31 ms) and DDM NpHR (0.44 ms). Such retardation in the transition from the P₃ to P₄ states was also encountered for the imposition of the negative membrane potential (18), supporting the repulsive interaction between the negative charge on the membrane surface and Cl⁻.

Another factor to retard the transition rate from O to NpHR′ embedded in the NL membrane would be interactions with Brub. A previous study reported that, in response to the Cl⁻ binding to or releasing from NpHR, the absorbance spectrum of Brub was changed, reflecting the altered electronic environment and/or distortion of its own configuration (18). Such spectral changes were detected in the transition from O to NpHR′ (18), suggesting specific interaction between Brub and NpHR. The more enhanced retardation was detected in MF NpHR because of the insufficient interaction of Brub with NpHR in NL-ND. The insufficient interaction of Brub with NpHR in NL-ND NpHR is supported by the minimal perturbation in the visible spectrum of Brub in the transition from N to O (Fig. 5 a), in contrast to those in the KM-1 membrane fragment (18). It is, therefore, plausible that loss of the specific interaction to induce the environmental perturbation around Brub in POPC-ND- and DDM NpHR accelerates the transition from O to NpHR′ in the photocycle of NpHR.

On the other hand, the effects of lipids on the time constant are more complicated in the transition from the P₄ to P₀ states (τ₄), corresponding to the transition from NpHR′ to NpHR. At the lower Cl⁻ (0.1 M) concentration, τ₄ of POPC-ND NpHR (17 ms) was shorter than that of NL-ND NpHR (41 ms), whereas the opposite effects were encountered for measurements at the higher Cl⁻ (1.0 M) concentration (59 and 27 ms for POPC-ND NpHR and NL-ND NpHR, respectively). By using vesicles from the KM-1 MF, the transition rate from NpHR′ to NpHR was reported to be reduced by the imposition of the negative membrane potential (18), reflecting the existence of the Cl⁻ binding site near the extracellular surface of the membrane. The slowdown of the transition from the P₄ to P₀ states observed for NL-ND NpHR at the low Cl⁻ concentration would, therefore, be due to the negatively charged membrane surface of the NL-reconstituted membrane. At the higher Cl⁻ concentration, however, the transition from the P₄ to P₀ states in NL-ND NpHR was accelerated, whereas the transition rate was retarded for POPC-ND NpHR. Such Cl⁻-concentration-dependent acceleration and deceleration of the transition rates from the P₄ to P₀ states was also detected for MF NpHR and DDM NpHR, respectively, suggesting that interactions with Brub at higher Cl⁻ concentration might accelerate the transition rate. As discussed above, Brub specifically interacts with NpHR in the Cl⁻-binding and releasing processes, and conformational changes of Brub associated with the transition from the P₄ to P₀ states were induced (18), but detailed Brub-mediated mechanisms for the regulation of the transition rate from the P₄ to P₀ states remain uncertain.

Although the kinetic property of NL-ND NpHR is similar to that of MF NpHR at high Cl⁻ concentration (1.0 M), the time constants of the transitions from the P₃ to P₄ states and from the P₄ to P₀ states under the diluted Cl⁻ concentration (0.1 M) were substantially shorter in NL-ND NpHR (τ₃, τ₄ = 8.0, 41 ms) when compared to those in MF NpHR (τ₃, τ₄ = 44, 237 ms). One possible reason for the short time constants in NL-ND NpHR would be the reduced content of Brub in the lipid bilayer of NL-NDs. The interactions of NpHR and Brub suppressed the conformational changes in NpHR as discussed above, and the low content of Brub in NL-ND NpHR is likely due to the washing out in the preparation of NL-NDs. Reduced Brub would result in fewer interactions of NpHR with Brub, resulting in a faster reaction rate for NL-ND NpHR.

As previously reported (17), conformational perturbation of the trimeric structure of NpHR induced the substantial changes in the photoreaction parameters. The mutation of Phe150, located at the central part of the NpHR trimer, to Tyr did not dissociate the NpHR trimer into the monomers, showing similar positive and negative CD bands in the visible region, but the time constant of the transition from the P₄ to P₀ states (τ₄) decreased to 20 ms (17). Although the CD band around 600 nm of NL-ND NpHR was similar to that of MF NpHR, indicating that the trimeric state of NpHR was not drastically perturbed by the reconstitution into NDs, the slight conformational difference of the trimeric states associated with the reconstitution into NDs would be one of the factors for the different photoreaction kinetic parameters between NL-ND NpHR and MF NpHR.

Another factor for the faster transition rates in NL-ND NpHR would be the lack of trimer-trimer interactions. Based on high-speed AFM images, “bipolar” cooperative effects (i.e., the coexistence of the positive and negative cooperativity effects) in decay kinetics are proposed to be mediated by trimer-trimer interactions. Under a relatively strong light illumination, early activated monomers within a trefoil decay more slowly, whereas the last monomer activated within a trefoil decays faster than in the case in which only one monomer within a trefoil is activated under weak light illumination (22). Because only one trimeric NpHR is reconstituted into NL-ND, no trefoil structure is formed. We postulate that decay of the light-activated monomer in NL-ND NpHR would, therefore, be faster than that of MF NpHR contained in a trefoil structure within the native membrane.

Conclusions

In this study, we have successfully reconstituted one trimeric NpHR into an ND using NLs as well as an artificial lipid, POPC. The spectroscopic and kinetic properties of NL-ND NpHR are rather similar to those of MF NpHR, but the Cl⁻ releasing and uptake of NL-ND NpHR were apparently different from those of MF NpHR, which is characterized by smaller K_d,N↔O, short time constants for the Cl⁻ uptake (τ₃), and faster recovering from the NpHR′ state (P₄) to the original state (P₀) (τ₄). Although POPC-ND NpHR using the lipid with the zwitterionic headgroup showed similar K_d,N↔O to that of NL-ND NpHR having negatively charged membrane surface, the Cl⁻ uptake from the extracellular side in the photocycle (τ₃) was accelerated, revealing that protein-lipid interactions are essential for the photoreaction cycle of NpHR. These functional differences are likely due to 1) the low content of Brub in the bilayer of NL-NDs, 2) the neutralization of the negative charges on the membrane surface of POPC-NDs, 3) the suppression of the conformational changes associated with Cl⁻ release by NpHR when reconstituted into NDs, 4) conformational perturbation in the trimeric state of NpHR, and 5) the lack of the intertrimer interactions of NpHR to decelerate the Cl⁻ uptake from the extracellular side. Given these results, we propose that membrane composition and trimer-trimer interactions are essential factors for regulating the Cl⁻-pumping function of NpHR.

Author Contributions

A.Y., T.T., K. Suzuki, E.H., and Y.K. performed experiments. A.Y., T.T., K. Suzuki, E.H., K. Shibasaki, and T.U. analyzed data. All the authors discussed the data. F.I., M.D., and K.I. designed research. A.Y., T.T., and K.I. wrote the article.

Editor: Sudha Chakrapani.