In bacteriorhodopsin (bR), Arg-82bR has been proven to be a very important residue for functional role of this light-driven proton pump. The arginine residue at this position is a super-conserved residue among archaeal rhodopsins. pharaonis phoborhodopsin (ppR; or called as "pharaonis sensory rhodopsin II") has its absorption maximum at 498 nm and acts as a sensor in the membrane of Natronobacterium pharaonis, mediating the negative phototaxis from the light of wavelength shorter than 520 nm. To investigate the role of the arginine residue (Arg-72ppR) of ppR corresponding to Arg-82bR, mutants whose Arg-72ppR was replaced by alanine (R72A), lysine (R72K), glutamine (R72Q) and serine (R72S) were prepared. These mutants were unstable in low concentrations of NaCl and lost their color gradually when the proteins were solubilized with 0.1% n-dodecyl-beta-D-maltoside. The order of instability was R72S > R72A > R72K > R72Q > the wild type. The rates of denaturation were reduced in a solution of high concentrations of monovalent anions.

Pharaonis phoborhodopsin (ppR) (also pharaonis sensory rhodopsin II) is a receptor of the negative phototaxis of Natronobacterium pharaonis. ppR forms a complex with its pharaonis halobacterial transducer (pHtrII), and this complex transmits the light signal to the sensory system in the cytoplasm. The expressed C-terminal-His tagged ppR and C-terminal-His tagged truncated pHtrII (t-Htr) in Escherichia coli (His means the 6x histidine tag) form a complex even in the presence of 0.1% of n-dodecyl-beta-D-maltoside, and the M-decay of the complex became about twice slower than that of ppR alone. The photocycling rates under varying concentration ratios of ppR to t-Htr in the presence of detergent were measured. The data were analyzed on the following assumptions: (1) the M-decay of both ppR alone and the complex followed a single exponential decay with different time constants; and (2) the M-decay under varying concentration ratios of ppR to t-Htr, therefore, followed a biexponential decay function which combined the decay of the free ppR and that of the complex as photoreactive species. From these analyses we estimated the dissociation constant (15.2 +/- 1.8 microM) and the number of binding sites (1.2 +/- 0.08).

A gene encoding putative retinal protein was cloned from Haloterrigena turkmenica (JCM9743). The deduced amino acid sequence was most closely related to that of deltarhodopsin, which functions as a light-driven H+ pump and was identified in a novel strain Haloterrigena sp. arg-4 (K. Ihara, T. Uemura, I. Katagiri, T. Kitajima-Ihara, Y. Sugiyama, Y. Kimura, Y. Mukohata, Evolution of the archaeal rhodopsins: Evolution rate changes by gene duplication and functional differentiation, J. Mol. Biol. 285 (1999) 163-174. GenBank Accession No. AB009620). Thus, we called the present protein H. turkmenica deltarhodopsin (HtdR) in this report. Differing from the Halobacterium salinarum bacteriorhodopsin (bR), functional expression of HtdR was achieved in Escherichia coli membrane with a high yield of 10-15 mg protein/L culture. The photocycle of purified HtdR was similar to that of bR. The photo-induced electrogenic proton pumping activity of HtdR was verified. We co-expressed both HtdR and EmrE, a proton-coupled multi-drug efflux transporter in E. coli, and the cells successfully extruded ethidium, a substrate of EmrE, on illumination.

Pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, psRII) is a receptor for negative phototaxis in Natronomonas pharaonis. In membranes, it forms a 2:2 complex with its transducer protein pHtrII, and the association is weakened by 2 orders of magnitude in the M intermediate (ppR(M)). Such a change is believed to correspond to the transfer of the light signal to pHtrII. A previous Fourier transform infrared (FTIR) study observed hydrogen-bonding alteration of Asn74 in pHtrII in the M state, suggesting a light-signaling pathway from the receptor to the transducer [Furutani, Y., Kamada, K., Sudo, Y., Shimono, K., Kamo, N., and Kandori, H. (2005) Biochemistry 44, 2909-2915]. In this paper, we measure temperature dependence of the ppR(M) minus ppR spectra in the absence and presence of pHtrII at 250-293 K. Significant temperature dependence was observed for the amide-I vibrations of helices only for the ppR/pHtrII complex, where the amplitude of amide-I vibrations was reduced at room temperature. (13)C-Labeling of ppR or pHtrII revealed that such spectral changes of helices originate from ppR and not pHtrII. The hydrogen-bonding alteration of Asn74 in pHtrII was temperature-independent, implying that the observed helical structural perturbation in ppR takes place in different region. On the other hand, temperature-dependent structural changes of helices were diminished for the complex of ppR with the G83C and G83F mutants of pHtrII. Gly83 is believed to connect the transmembrane helix and cytosolic linker region in a flexible kink near the membrane surface of pHtrII, and its replacement by Cys or Phe abolishes the photosensory function. The present study provides direct experimental evidence that Gly83 plays an important structural role in the activation processes of the ppR/pHtrII complex. A molecular mechanism of protein structural changes in the ppR/pHtrII complex is discussed on the basis of the present FTIR results.

aIms: There has been a considerable increase in the number of seizures of amphetamine-type stimulant (ATS) tablets in Japan. The ATS tablets contain one or more of active ingredients, which include 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxyethylamphetamine (MDEA), methamphetamine (MA), etc.. In addition, the tablets often contain other components with hallucinogenic and/or stimulant effects such as ketamine and caffeine. various components in such tablets might interact in the body, leading to serious toxic symptoms. Therefore, the prediction of interactions among drugs is important for public health and forensic analysis. We examined the interaction among the components in ATS tablets using Caco-2 cells derived from human colons and using rats in order to investigate the interactions in absorption process on digestive tract that was the first step of pharmacokinetics after oral intake.

Four rhodopsins, bacteriorhodopsin (bR), halorhodopsin (hR), sensory rhodopsin (sR) and phoborhodopsin (pR) exist in archaeal membranes. bR and hR work as a light-driven ion pump. sR and pR work as a photo-sensor of phototaxis, and form signaling complexes in membranes with their respective cognate transducer proteins HtrI (with sR) and HtrII (with pR), through which light signals are transmitted to the cytoplasm. What is the determining factor(s) of the specific binding to form the complex? Binding of the wild-type or mutated rhodopsins with HtrII was measured by isothermal titration calorimetric analysis (ITC). bR and hR could not bind with HtrII. On the other hand, sR could bind to HtrII, although the dissociation constant (K(D)) was about 100 times larger than that of pR. An X-ray crystallographic structure of the pR/HtrII complex revealed formation of two specific hydrogen bonds whose pairs are Tyr199(pR)/Asn74(HtrII) and Thr189(pR)/Glu43(HtrII)/Ser62(HtrII). To investigate the importance of these hydrogen bonds, the K(D) value for the binding of various mutants of bR, hR, sR and pR with HtrII was estimated by ITC. The K(D) value of T189V(pR)/Y199F(pR), double mutant/HtrII complex, was about 100-fold larger than that of the wild-type pR, whose K(D) value was 0.16 microM. On the other hand, bR and hR double mutants, P200T(bR)/V210Y(bR) and P240T(hR)/F250Y(hR), were able to bind with HtrII. The K(D) value of these complexes was estimated to be 60.1(+/-10.7) microM for bR and to be 29.1(+/-6.1) microM for hR, while the wild-type bR and hR did not bind with HtrII. We concluded that these two specific hydrogen bonds play important roles in the binding between the rhodopsins and transducer protein.

Natronomonas (Natronobacterium) pharaonis halorhodopsin (NpHR) is an inward light-driven Cl- ion pump. For efficient Cl- transport, the existence of Cl--binding or -interacting sites in both extracellular (EC) and cytoplasmic (CP) channels is postulated. Candidates include Arg123 and Thr126 in EC channels and Lys215 and Thr218 in CP channels. The roles played by these amino acid residues in anion binding and in the photocycle have been investigated by mutation of the amino acid residues at these positions. Anion binding was assayed by changes in circular dichroism and the shift in the absorption maximum upon addition of Cl- to anion-free NpHR. The binding affinity was affected in mutants in which certain EC residues had been replaced; this finding revealed the importance of Arg123. On the other hand, mutants in which certain residues in the CP channel were replaced (CP mutants) did not show changes in their dissociation constants. The photocycles of these mutants were also examined, and in the case of the EC mutants, the transition to the last step was greatly delayed; on the other hand, in the CP mutants, L2-photointermediate decay was significantly prolonged, except in the case of K215Q, which lacked the O-photointermediate. The importance of Thr218 for binding of Cl- to the CP channel was indicated by these results. On the basis of these observations, the possible anion transport mechanism of NpHR was discussed.

Natronomonas (Natronobacterium) pharaonis halorhodopsin (NpHR) is a transmembrane, seven-helix retinal protein of the archaeal bacterium and acts as an inward light-driven chloride ion pump in the membrane. The denaturation process of NpHR solubilized with n-octyl-beta-d-glucopyranoside (OG) was investigated to clarify the effects of the chloride ion and pH on the stability and bleaching of the NpHR chromophore. Initially, active NpHR solubilized with n-dodecyl-beta-d-maltopyranoside (DM) was obtained from the recombinant halo-opsin (NpHO), which was expressed in Escherichia coli cells, by adding all-trans retinal to the medium. Apparent molecular weight of the active NpHR solubilized with DM, which was determined by gel-filtration chromatography and dynamic light scattering, indicated the oligomeric state. The bleaching of NpHR in the dark by the addition of 50 mM OG in the presence and absence of chloride was investigated. In the presence of 256 mM NaCl, the bleaching of NpHR was strongly inhibited. On the other hand, in the absence of NaCl, an immediate decrease of absorbance at 600 nm was observed. Stopped-flow rapid-mixing analysis clarified the bleaching process in the absence of chloride as DM-NpHR (oligomeric) <--> OG-NpHR (disassembled) <--> intermediate --> NpHO and free retinal, and each rate constant were determined. The formation of an intermediate (450 nm) in the dark was found to be strongly dependent on pH, as well as anion and detergent concentrations. The disassembling and protonation of a Schiff base corresponding to the bleaching intermediate is also discussed.

In visual and archaeal rhodopsins, light energy is stored in the chromophore-protein interaction after retinal photoisomerization. This paper reports a novel method to monitor the steric constraint after retinal isomerization by use of enhanced C-D stretching vibrations. In the difference FTIR spectra between an archaeal light-sensor pharaonis phoborhodopsin (ppR) and the primary K intermediate at 77 K, no peaks were observed in the 2160-2330 cm-1 region for deuterated retinals at position 7, 8, 10, 11, 12, and 15, whereas a strong peak appeared at 2244 cm-1 for the K intermediate of ppR possessing a C14-D-labeled retinal. The 2244-cm-1 band is assigned as the C14-D stretching vibration, and enhanced absorption in the K state probably originates from the local steric constraint at the C14-D position (also possible electrostatic field effects) after the C13=C14 double bond rotation.

pHtrII, a pharaonis halobacterial transducer protein, possesses two transmembrane helices and forms a signaling complex with pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, NpSRII) within the halobacterial membrane. This complex transmits a light signal to the sensory system located in the cytoplasm. It has been suggested that the linker region connecting the transmembrane region and the methylation region of pHtrII is important for binding to ppR and subsequent photosignal transduction. In this study, we present evidence to suggest that the linker region itself interacts directly with ppR in addition to the interaction in the membrane region. An in vitro pull-down assay revealed that the linker region bound to ppR, and its dissociation constant (K(D)) was estimated to be approximately 10 microM using isothermal titration calorimetry (ITC). Solution NMR analyses showed that ppR interacted with the linker region of pHtrII (pHtrII(G83)(-)(Q149)) and resulted in the broadening of many peaks, indicating structural changes within this region. These results suggest that the pHtrII linker region interacts directly with ppR. There was no demonstrable interaction between the C-terminal region of ppR (ppR(Gly224)(-)(His247)) and either the linker region (pHtrII(G83)(-)(Q149)) or the transmembrane region (pHtrII(M1)(-)(E114)) of pHtrII. On the basis of the NMR, CD, and photochemical data, we discuss the structural changes and role of the linker region of pHtrII in relation to photosignal transduction.

In the present study, we measured an electric current induced by substrate transport in a HeLa cell over-expressing a human intestinal di/tri-peptide transporter using the whole-cell patch-clamp technique. Gly-Sar, a typical substrate, induced an inward current associated with its uptake, which showed concentration-dependency following Michaelis-Menten-type kinetics with an apparent K(0.5) of 1.3mM as well as voltage-dependency. An outward current accompanying the efflux of Gly-Sar was also observed after washing out the cell. This outward current was voltage-dependent and was reduced by the inward proton gradient. In the case of hydrophobic dipeptides such as Gly-Phe and Gly-Leu, a distinctive current was observed: after washing out the cells, no outward current was observed, but rather, an 'inward leak' current was sustained in spite of the absence of transportable substrate. This leaky current was abolished by the perfusion of Gly-Sar and subsequent washing. It is considered that the hydrophobic substrate sticks within the substrate-binding site and causes the newly observed state, or the 'inward leak' current.

Proteorhodopsin, a retinal protein of marine proteobacteria similar to bacteriorhodopsin of the archaea, is a light-driven proton pump. Absorption of a light quantum initiates a reaction cycle (turnover time of ca. 50 ms), which includes photoisomerization of the retinal from the all-trans to the 13-cis form and transient deprotonation of the retinal Schiff base, followed by recovery of the initial state. We report here that in addition to this fast cyclic conversion, illumination at high pH results in accumulation of a long-lived photoproduct absorbing at 362 nm. This photoconversion is much more efficient in the D227N mutant in which the anionic Asp227, which together with Asp97 constitutes the Schiff base counterion, is replaced with a neutral residue. Upon illumination at pH 8.5, most of the D227N pigment is converted to the 362 nm species, with a quantum efficiency of ca. 0.2. The pK(a) for this transition in the wild type is 9.6, but decreased to 7.5 after mutation of Asp227. The short wavelength of the absorption maximum of the photoproduct indicates that it has a deprotonated Schiff base. In the dark, this photoproduct is converted back to the initial pigment with a time constant of 30 min (in D227N, at pH 8.5), but it can be reconverted more rapidly by illumination with near-UV light. Experiments with "locked" retinal analogues which selectively exclude rotation around either the C9=C10, C11=C12, or C13=C14 bond show that formation of the 362 nm species involves isomerization around the C13=C14 bond. In agreement with this, retinal extraction indicates that the 362 nm photoproduct is 13-cis whereas the initial state is predominantly all-trans. A rapid shift of the pH from 8.5 to 4 greatly accelerates thermal reconversion of the 362 nm species to the initial pigment, suggesting that its recovery involving the thermal isomerization of the chromophore is controlled by ionizable residues, primarily the Schiff base and Asp97. The transformation to the long-lived 362 nm photoproduct is apparently a side reaction of the photocycle, a response to high pH, caused by alteration of the normal reprotonation and reisomerization pathway of the Schiff base.

Halorhodopsin is a light-driven chloride ion pump. Chloride ion is bound in the Schiff base region of the retinal chromophore, and unidirectional chloride transport is probably enforced by the specific hydrogen-bonding interaction with the protonated Schiff base and internal water molecules. In this article, we study hydrogen-bonding alterations of the Schiff base and water molecules in halorhodopsin of Natronobacterium pharaonis (pHR) by assigning their N-D and O-D stretching vibrations in D(2)O, respectively. Highly accurate low-temperature Fourier transform infrared spectroscopy revealed that hydrogen bonds of the Schiff base and water molecules are weak in the unphotolyzed state, whereas they are strengthened upon retinal photoisomerization. Halide dependence of the stretching vibrations enabled us to conclude that the Schiff base forms a direct hydrogen bond with Cl(-) only in the K intermediate. Hydrogen bond of the Schiff base is further strengthened in the L(1) intermediate, whereas the halide dependence revealed that the acceptor is not Cl(-), but presumably a water molecule. Thus, it is concluded that the hydrogen-bonding interaction between the Schiff base and Cl(-) is not a driving force of the motion of Cl(-). Rather, the removal of its hydrogen bonds with the Schiff base and water(s) makes the environment around Cl(-) less polar in the L(1) intermediate, which presumably drives the motion of Cl(-) from its binding site to the cytoplasmic domain.

We have recorded 13C NMR spectra of the [3-13C]Ala, [1-13C]Val-labeled pharaonis transducer pHtrII(1–159) in the presence and absence of phoborhodopsin (ppR or sensory rhodopsin II) in egg phosphatidylcholine or dimyristoylphosphatidylcholine bilayers by means of site-directed (amino acid specific) solid-state NMR. Two kinds of 13C NMR signals of [3-13C]Ala-pHtrII complexed with ppR were clearly seen with dipolar decoupled magic angle spinning (DD-MAS) NMR. One of these resonances was at the peak position of the low-field α-helical peaks (αII-helix) and is identified with cytoplasmic α-helices protruding from the bilayers; the other was the high-field α-helical peak (αI-helix) and is identified with the transmembrane α-helices. The first peaks, however, were almost completely suppressed by cross-polarization magic angle spinning (CP-MAS) regardless of the presence or absence of ppR or by DD-MAS NMR in the absence of ppR. This is caused by an increased fluctuation frequency of the cytoplasmic α-helix from 105 Hz in the uncomplexed states to >106 Hz in the complexed states, leading to the appearance of peaks that were suppressed because of the interference of the fluctuation frequency with the frequency of proton decoupling (105 Hz), as viewed from the 13C NMR spectra of [3-13C]Ala-labeled pHtrII. Consistent with this view, the 13C DD-MAS NMR signals of the cytoplasmic α-helices of the complexed [3-13C]Ala-pHtrII in the dimyristoylphosphatidylcholine (DMPC) bilayer were partially suppressed at 0°C due to a decreased fluctuation frequency at the low temperature. In contrast, examination of the 13C CP-MAS spectra of [1-13C]Val-labeled complexed pHtrII showed that the 13C NMR signals of the transmembrane α-helix were substantially suppressed. These spectral changes are again interpreted in terms of the increased fluctuation frequency of the transmembrane α-helices from 103 Hz of the uncomplexed states to 104 Hz of the complexed states. These findings substantiate the view that the transducers alone are in an aggregated or clustered state but the ppR-pHtrII complex is not aggregated. We show that 13C NMR is a very useful tool for achieving a better understanding of membrane proteins which will serve to clarify the molecular mechanism of signal transduction in this system.

The interaction of lipophilic cations, tetraphenylphosphonium and triphenylphosphonium homologues with liposomes was investigated using immobilized liposome chromatography (ILC). Large unilamellar liposomes with a mean diameter of 100 nm were stably immobilized in chromatographic gel beads by avidin–biotin. The distribution coefficient calculated from (Ve−V0)/Vs (Ve, retention volume; V0, the void volume; Vs, the stationary phase volume) was found to be independent of flow rate, injection amount and gel bed volume, which is consistent with chromatograph theory. The relationship between the bandwidth and solvent flow rate did not follow band-broadening theories reported thus far. We hypothesized that the solvent might be forced to produce large eddies, spirals or turbulent flow due to the presence of liposomes fixed in the gel. Therefore, we developed a new theory for ILC elution: The column is composed of a number of thin disks containing liposomes and solution, and within each disk the solution is well mixed. This theory accounts for our results, and we were able to use it to estimate the rate constants of association and dissociation of the phosphonium to/from liposomes.

Pharaonis phoborhodopsin (ppR, or pharaonis sensory rhodopsin II, NpsRII) is a sensor for the negative phototaxis of Natronomonas (Natronobacterium) pharaonis. Arginine 72 of ppR corresponds to Arg-82 of bacteriorhodopsin, which is a highly conserved residue among microbial rhodopsins. Using various Arg-72 ppR mutants, we obtained the following results: 1), Arg-72ppR together possibly with Asp-193 influenced the pKa of the counterion of the protonated Schiff base. 2), The M-rise became approximately four times faster than the wild-type. 3), Illumination causes proton uptake and release, and the pH profiles of the sequence of these two proton movements were different between R72A mutant and the wild-type; it is inferred that Arg-72 connects the proton transfer events occurring at both the Schiff base and an extracellular proton-releasing residue (Asp-193). 4), The M-decays of Arg-72 mutants were faster (∼8–27 folds at pH 8 depending on mutants) than the wild-type, implying that the guanidinium prevents the proton transfer from the extracellular space to the deprotonated Schiff base. 5), The proton-pumping activities were decreased for mutants having increased M-decay rates, but the extent of the decrease was smaller than expected. The role of Arg-72 of ppR on the photochemistry was discussed.

pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, psRII) is a photo-receptor for negative phototaxis in Natronobacterium pharaonis. During the photoreaction cycle (photocycle), ppR exhibits intraprotein proton movements, resulting in proton pumping from the cytoplasmic to the extracellular side, although it is weak. In this study, light-induced proton uptake and release of ppR reconstituted with phospholipid were analyzed using a SnO(2) electrode. The reconstituted ppR exhibited properties in proton uptake and release that are different from those of dodecyl maltoside solubilized samples. It showed fast proton release before the decay of ppR(M) (M-photointermediate) followed by proton uptake, which was similar to that of bacteriorhodopsin (BR), a light-driven proton pump. Mutant analysis assigned Asp193 to one (major) of the members of the proton-releasing group (PRG). Fast proton release was observed only when the pH was approximately 5-8 in the presence of Cl(-). When Cl(-) was replaced with SO(4)(2-), the reconstituted ppR did not exhibit fast proton release at any pH, suggesting Cl(-) binding around PRG. PRG in BR consists of Glu204 (Asp193 in ppR) and Glu194 (Pro183 in ppR). Replacement of Pro183 by Glu/Asp, a negatively charged residue, led to Cl(-)-independent fast proton release. The transducer binding affected the properties of PRG in ppR in the ground state and in the ppR(M) state, suggesting that interaction with the transducer extends to the extracellular surface of ppR. Differences and similarities in the molecular mechanism of the proton movement between ppR and BR are discussed.