Transducer protein HtrI controls proton movements in sensory rhodopsin I

Sensory rhodopsin I (SR-I lambda(max) 587 nm) is a phototaxis receptor in the archaeon Halobacterium salinarium. Photoisomerization of retinal in SR-I generates a long-lived intermediate with lambda(max) 373 nm which transmits a signal to the membrane-bound transducer protein HtrI. Although SR-I is structurally similar to the electrogenic proton pump bacteriorhodopsin (BR), early studies showed its photoreactions do not pump protons, nor result in membrane hyperpolarization. These studies used functionally active SR-I, that is, SR-I complexed with its transducer HtrI. Using recombinant DNA methods we have expressed SR-I protein containing mutations in ionizable residues near the protonated Schiff base, and studied wild-type and site-specifically mutated SR-I in the presence and absence of the transducer protein. UV-Vis kinetic absorption spectroscopy, FT-IR, and pH and membrane potential probes reveal transducer-free SR-I photoreactions result in vectorial proton translocation across the membrane in the same direction as that of BR. This proton pumping is suppressed by interaction with transducer which diverts the proton movements into an electroneutral path. A key step in this diversion is that transducer interaction raises the pK(a) of the aspartyl residue in SR-I (Asp76) which corresponds to the primary proton-accepting residue in the BR pump (Asp85). In transducer-free SR-I, our evidence indicates the pK(a) of Asp76 is 7.2, and ionized Asp76 functions as the Schiff base proton acceptor in the SR-I pump. In the SR-I/HtrI complex, the pK(a) of Asp76 is 8.5, and therefore at physiological pH (7.4) Asp76 is neutral. Protonation changes on Asp76 are clearly not required for signaling since the SR-I mutants D76N and D76A are active in phototaxis. The latent proton-translocation potential of SR-I may reflect the evolution of the SR-I sensory signaling mechanism from the proton pumping mechanism of BR.     

Variations on a molecular switch: transport and sensory signalling by archaeal rhodopsins

The archaeal rhodopsins are a family of seven-transmembrane-helix, visual pigment-like proteins found in Halobacterium salinarum and related halophilic Archaea. Two, bacteriorhodopsin (BR) and halorhodopsin (HR), are transport rhodopsins that carry out light-driven electrogenic translocation of protons and chloride, respectively, across the cell membrane. The other two, sensory rhodopsins I and II (SRI and SRII), are phototaxis receptors that send signals to tightly bound transducer proteins that in turn control a phosphorylation cascade modulating the cell's flagellar motors. Recent progress has cast light on how nature has modified the common design of these proteins to carry out their distinctly different functions: electrogenic ion transport and non-electrogenic signal transduction. A key shared mechanism between BR and SRII appears to be an interhelical salt bridge locked conformational switch that is released by photoisomerization of retinal. In BR disruption of the lock opens a cytoplasmic half-channel that ensures uptake of the transported proton from the cytoplasmic side of the membrane at a critical time in the pumping cycle. Transducer-free SRI uses the same mechanism to carry out light-driven proton transport, but interaction with its transducer blocks the cytoplasmic half-channel thereby interrupting the transport cycle. In SRI, transducer interaction also disrupts the salt bridge in the dark, poising the receptor in an intermediate conformation able to produce opposite signals depending on the colour of the stimulus light. A model for signalling is proposed in which the salt bridge-controlled half-channel is used to modulate interaction with the Htr proteins when the receptor signalling states are formed.     

Constitutive activity in chimeras and deletions localize sensory rhodopsin II/HtrII signal relay to the membrane-inserted domain

Halobacterium salinarum sensory rhodopsin II (HsSRII) is a phototaxis receptor for blue-light avoidance that relays signals to its tightly bound transducer HsHtrII (H. salinarum haloarchaeal transducer for SRII). We found that disruption of the salt bridge between the protonated Schiff base of the receptor's retinylidene chromophore and its counterion Asp73 by residue substitutions D73A, N or Q constitutively activates HsSRII, whereas the corresponding Asp75 counterion substitutions do not constitutively activate Natronomonas pharaonis SRII (NpSRII) when complexed with N. pharaonis haloarchaeal transducer for SRII (NpHtrII). However, NpSRII(D75Q) in complex with HsHtrII is fully constitutively active, showing that transducer sensitivity to the receptor signal contributes to the phenotype. The swimming behaviour of cells expressing chimeras exchanging portions of the two homologous transducers localizes their differing sensitivities to the HtrII transmembrane domains. Furthermore, deletion constructs show that the known contact region in the cytoplasmic domain of the NpSRII-NpHtrII complex is not required for phototaxis, excluding the domain as a site for signal transmission. These results distinguish between the prevailing models for SRII-HtrII signal relay, strongly supporting the 'steric trigger-transmembrane relay model', which proposes that retinal isomerization directly signals HtrII through the mid-membrane SRII-HtrII interface, and refuting alternative models that propose signal relay in the cytoplasmic membrane-proximal domain.     

Proton transfer reactions in the F86D and F86E mutants of pharaonis phoborhodopsin (sensory rhodopsin II)

pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, psRII), a negative phototaxis receptor of Natronobacterium pharaonis, can use light to pump a proton in the absence of its transducer protein. However, the pump activity is much lower than that of the light-driven proton-pump bacteriorhodopsin (BR). ppR's pump activity is known to be increased in a mutant protein, in which Phe86 is replaced with Asp (F86D). Phe86 is the amino acid residue corresponding to Asp96 in BR, and we expect that Asp86 plays an important role in the proton transfer at the highly hydrophobic cytoplasmic domain of the F86D mutant ppR. In this article, we studied protein structural changes and proton transfer reactions during the photocycles of the F86D and F86E mutants in ppR by means of Fourier transform infrared (FTIR) spectroscopy and photoelectrochemical measurements using a tin oxide (SnO2) electrode. FTIR spectra of the unphotolyzed state and the K and M intermediates are very similar among F86D, F86E, and the wild type. Asp86 or Glu86 is protonated in F86D or F86E, respectively, and the pK(a) > 9. During the photocycle, the pK(a) is lowered and deprotonation of Asp86 or Glu86 is observed. Detection of both deprotonation of Asp86 or Glu86 and concomitant reprotonation of the 13-cis chromophore implies the presence of a proton channel between position 86 and the Schiff base. However, the photoelectrochemical measurements revealed proton release presumably from Asp86 or Glu86 to the cytoplasmic aqueous phase in the M state. This indicates that the ppR mutants do not have the BR-like mechanism that conducts a proton uniquely from Asp86 or Glu86 (Asp96 in BR) to the Schiff base, which is possible in BR by stepwise protein structural changes at the cytoplasmic side. In ppR, there is a single open structure at the cytoplasmic side (the M-like structure), which is shown by the lack of the N-like protein structure even in F86D and F86E at alkaline pH. Therefore, it is likely that a proton can be conducted in either direction, the Schiff base or the bulk, in the open M-like structure of F86D and F86E.     

Five residues in the HtrI transducer membrane-proximal domain close the cytoplasmic proton-conducting channel of sensory rhodopsin I

Transducer-free sensory rhodopsins carry out light-driven proton transport in Halobacterium salinarum membranes. Transducer binding converts the proton pumps to signal-relay devices in which the transport is inhibited. In sensory rhodopsin I (SRI) binding of its cognate transducer HtrI inhibits transport by closing a cytoplasmic proton-conducting channel necessary for proton uptake during the SRI photochemical reaction cycle. To investigate the channel closure, a series of HtrI mutants truncated in the membrane-proximal cytoplasmic portion of an SRI-HtrI fusion were constructed and expressed in H. salinarum membranes. We found that binding of the membrane-embedded portion of HtrI is insufficient for channel closure, whereas cytoplasmic extension of the second HtrI transmembrane helix by 13 residues blocks proton conduction through the channel as well as full-length HtrI. Specifically the closure activity is localized in this 13-residue membrane-proximal cytoplasmic domain to the 5 final residues, each of which incrementally contributes to reduction of proton conductivity. Moreover, these same residues in the dark incrementally and proportionally increase the pKa of the Asp-76 counterion to the protonated Schiff base chromophore in the membrane-embedded photoactive site. We conclude that this critical region of HtrI alters the dark conformation of SRI as well as light-induced channel opening. The 5 residues in HtrI correspond in position to 5 residues demonstrated on the homologous NpHtrII to interact with the E-F loop of its cognate receptor NpSRII in the accompanying article (Yang, C.-S., Sineshchekov, O., Spudich, E. N., and Spudich, J. L. (2004) J. Biol. Chem. 279, 42970-42976). These results strongly suggest that the membrane-proximal region of Htr proteins interact with their cognate sensory rhodopsin cytoplasmic domains as part of the signal-relay coupling between the proteins.     

The signal transfer from the receptor NpSRII to the transducer NpHtrII is not hampered by the D75N mutation

Sensory rhodopsin II (NpSRII) is a phototaxis receptor of Natronomonas pharaonis that performs its function in complex with its cognate transducer (NpHtrII). Upon light activation NpSRII triggers by means of NpHtrII a signal transduction chain homologous to the two component system in eubacterial chemotaxis. The D75N mutant of NpSRII, which lacks the blue-shifted M intermediate and therefore exhibits a significantly faster photocycle compared to the wild-type, mediates normal phototaxis responses demonstrating that deprotonation of the Schiff base is not a prerequisite for transducer activation. Using site-directed spin labeling and time resolved electron paramagnetic-resonance spectroscopy, we show that the mechanism revealed for activation of the wild-type complex, namely an outward tilt motion of the cytoplasmic part of the receptor helix F and a concomitant rotation of the transmembrane transducer helix TM2, is also valid for the D75N variant. Apparently, the D75N mutation shifts the ground state conformation of NpSRII-D75N and its cognate transducer into the direction of the signaling state.     

Alteration of conformation and dynamics of bacteriorhodopsin induced by protonation of Asp 85 and deprotonation of Schiff base as studied by 13C NMR

According to previous X-ray diffraction studies, the D85N mutant of bacteriorhodopsin (bR) with unprotonated Schiff base assumes a protein conformation similar to that in the M photointermediate. We recorded (13)C NMR spectra of [3-(13)C]Ala- and [1-(13)C]Val-labeled D85N and D85N/D96N mutants at ambient temperature to examine how conformation and dynamics of the protein backbone are altered when the Schiff base is protonated (at pH 7) and unprotonated (at pH 10). Most notably, we found that the peak intensities of three to four [3-(13)C]Ala-labeled residues from the transmembrane alpha-helices, including Ala 39, 51, and 53 (helix B) and 215 (helix G), were suppressed in D85N and D85N/D96N both from CP-MAS (cross polarization-magic angle spinning) and DD-MAS (dipolar decoupled-magic angle spinning) spectra, irrespective of the pH. This is due to conformational change and subsequent acquisition of intermediate time-range motions, with correlation times in the order of 10(-)(5) or 10(-)(4) s, which interferes with proton decoupling frequency or frequency of magic angle spinning, respectively, essential for an attempted peak-narrowing to achieve high-resolution NMR signals. Greater changes were achieved, however, at pH 10, which indicate large-amplitude motions of transmembrane helices upon deprotonation of Schiff base and the formation of the M-like state in the absence of illumination. The spectra detected more rapid motions in the extracellular and/or cytoplasmic loops, with correlation times increasing from 10(-)(4) to 10(-)(5) s. Conformational changes in the transmembrane helices were located at helices B, G, and D as viewed from the above-mentioned spectral changes, as well as at 1-(13)C-labeled Val 49 (helix B), 69 (B-C loop), and [3-(13)C]Ala-labeled Ala 126 (D-helix) signals, in addition to the cytoplasmic and extracellular loops. Further, we found that in the M-like state the charged state of Asp 96 at the cytoplasmic side substantially modulated the conformation and dynamics of the extracellular region through long-distance interaction.     

Conformational changes in the photocycle of Anabaena sensory rhodopsin: absence of the Schiff base counterion protonation signal

Anabaena sensory rhodopsin (ASR) is a novel microbial rhodopsin recently discovered in the freshwater cyanobacterium Anabaena sp. PCC7120. This protein most likely functions as a photosensory receptor as do the related haloarchaeal sensory rhodopsins. However, unlike the archaeal pigments, which are tightly bound to their cognate membrane-embedded transducers, ASR interacts with a soluble cytoplasmic protein analogous to transducers of animal vertebrate rhodopsins. In this study, infrared spectroscopy was used to examine the molecular mechanism of photoactivation in ASR. Light adaptation of the pigment leads to a phototransformation of an all-trans/15-anti to 13-cis/15-syn retinylidene-containing species very similar in chromophore structural changes to those caused by dark adaptation in bacteriorhodopsin. Following 532 nm laser-pulsed excitation, the protein exhibits predominantly an all-trans retinylidene photocycle containing a deprotonated Schiff base species similar to those of other microbial rhodopsins such as bacteriorhodopsin, sensory rhodopsin II, and Neurospora rhodopsin. However, no changes are observed in the Schiff base counterion Asp-75, which remains unprotonated throughout the photocycle. This result along with other evidence indicates that the Schiff base proton release mechanism differs significantly from that of other known microbial rhodopsins, possibly because of the absence of a second carboxylate group at the ASR photoactive site. Several conformational changes are detected during the ASR photocycle including in the transmembrane helices E and G as indicated by hydrogen-bonding alterations of their native cysteine residues. In addition, similarly to animal vertebrate rhodopsin, perturbations of the polar head groups of lipid molecules are detected.     

Proteorhodopsin is a light-driven proton pump with variable vectoriality

Proteorhodopsin, a homologue of archaeal bacteriorhodopsin (BR), belongs to a newly identified family of retinal proteins from marine bacteria, which could play an important role in the energy balance of the biosphere. We cloned the cDNA sequence of proteorhodopsin by chemical gene synthesis, expressed the protein in Escherichia coli cells, purified and reconstituted the protein in its functional active state. The photocycle characteristics were determined by time-resolved absorption and Fourier transform infrared (FT-IR) spectroscopy. The pH-dependence of the absorption spectrum indicates that the pK(a) of the primary acceptor of the Schiff base proton (Asp97) is 7.68. Generally, the photocycle of proteorhodopsin is similar to that of BR, although an L-like photocycle intermediate was not detectable. Whereas at pH>7 an M-like intermediate is formed upon illumination, at pH 5 no M-like intermediate could be detected. As the photocycle kinetics do not change between the acidic and alkaline state of proteorhodopsin, the only difference between these two forms is the protonation status of Asp97. This is corroborated by time-resolved FT-IR spectroscopy, which demonstrates that proton transfer from the retinal Schiff base to Asp97 is observed at alkaline pH, but the other vibrational changes are essentially pH-independent.After reconstitution into proteoliposomes, light-induced proton currents of proteorhodopsin were measured in a compound membrane system where proteoliposomes were adsorbed to planar lipid bilayers. Our results show that proteorhodopsin is a light-driven proton pump with characteristics similar to those of BR at alkaline pH. However, at acidic pH, the direction of proton pumping is inverted. Complementary experiments were carried out on proteorhodopsin expressed heterologously in Xenopus laevis oocytes under voltage clamp conditions.The following results were obtained. (1) At alkaline pH, proteorhodopsin mediates outwardly directed proton pumping like BR. (2) The direction of proton pumping can be inverted, when Asp97 is protonated. (3) The current can be inverted by changes of the polarity of the applied voltage. (4) The light intensity-dependence of the photocurrents leads to the conclusion that the alkaline form of proteorhodopsin shows efficient proton pumping after sequential excitation by two photons.     

Probing the sensory rhodopsin II binding domain of its cognate transducer by calorimetry and electrophysiology

Sensory rhodopsin II, a repellent phototaxis receptor from Natronobacterium pharaonis (NpSRII) forms a tight complex with its cognate transducer (NpHtrII). Light excitation of the receptor triggers conformational changes in both proteins, thereby activating the cellular two-component signalling cascade. In membranes, the two proteins form a 2:2 complex, which dissociates to a 1:1 heterodimer in micelles. Complexed to the transducer sensory rhodopsin II is no longer capable of light-driven proton pumping. In order to elucidate the dimerisation and the size of the receptor-binding domain of the transducer, isothermal titration calorimetry and electrophysiological experiments have been carried out. It is shown, that an N-terminal sequence of 114 amino acid residues is sufficient for tight binding (K(d)=240nM; DeltaH=-17.6kJmol(-1)) and for inhibiting the proton transfer. These data and results obtained from selected site-directed mutants indicate a synergistic interplay of transducer transmembrane domain (1-82) and cytoplasmic peptide (83-114) leading to an optimal and specific interaction between receptor and transducer.     

FTIR study of the L intermediate of Anabaena sensory rhodopsin: structural changes in the cytoplasmic region

Anabaena sensory rhodopsin (ASR) is an archaeal-type rhodopsin found in eubacteria. The gene encoding ASR forms a single operon with ASRT (ASR transducer) that is a 14 kDa soluble protein, suggesting that ASR functions as a photochromic sensor by activating the soluble transducer. One of the characteristics of ASR is that the formation of the M intermediate accompanies a proton transfer from the Schiff base to Asp217 in the cytoplasmic side [Shi, L., Yoon, S. R., Bezerra, A. G., Jr., Jung, K. H., and Brown, L. S. (2006) J. Mol. Biol. 358, 686-700], in remarkable contrast to other archaeal-type rhodopsins such as a light-driven proton-pump, bacteriorhodopsin (BR). In this study, we applied low-temperature Fourier transform infrared (FTIR) spectroscopy to the all- trans form of ASR at 170 K, and compared the structural changes in the L intermediate with those of BR. The ASR L minus ASR difference spectra were essentially similar to those for BR, suggesting common structures for the L state in ASR and BR. On the other hand, unique CO stretching bands of a protonated carboxylic acid were observed at 1722 (+) and 1703 (-) cm (-1) at pH 5 and 7, and assigned to Glu36 by use of mutants. Glu36 is located at the cytoplasmic side, and the distance from the Schiff base is about 20 A. This result shows the structural changes at the cytoplasmic surface in ASR L. pH-dependent frequency change was also observed for a water stretching vibration, suggesting that the water molecule is involved in a hydrogen-bonding network with Glu36 and Asp217. Unique hydrogen-bonding network in the cytoplasmic domain of ASR will be discussed.     

Hydration switch model for the proton transfer in the Schiff base region of bacteriorhodopsin

In a light-driven proton-pump protein, bacteriorhodopsin (BR), protonated Schiff base of the retinal chromophore and Asp85 form ion-pair state, which is stabilized by a bridged water molecule. After light absorption, all-trans to 13-cis photoisomerization takes place, followed by the primary proton transfer from the Schiff base to Asp85 that triggers sequential proton transfer reactions for the pump. Fourier transform infrared (FTIR) spectroscopy first observed O-H stretching vibrations of water during the photocycle of BR, and accurate spectral acquisition has extended the water stretching frequencies into the entire stretching frequency region in D(2)O. This enabled to capture the water molecules hydrating with negative charges, and we have identified the water O-D stretch at 2171 cm(-1) as the bridged water interacting with Asp85. We found that retinal isomerization weakens the hydrogen bond in the K intermediate, but not in the later intermediates such as L, M, and N. On the basis of the observation particularly on the M intermediate, we proposed a model for the mechanism of proton transfer from the Schiff base to Asp85. In the "hydration switch model", hydration of a water molecule is switched in the M intermediate from Asp85 to Asp212. This will have raised the pK(a) of the proton acceptor, and the proton transfer is from the Schiff base to Asp85.     

FTIR study of the retinal Schiff base and internal water molecules of proteorhodopsin

Proteorhodopsin (PR), an archaeal-type rhodopsin found in marine bacteria, is a light-driven proton pump similar to bacteriorhodopsin (BR). It is known that Asp97, a counterion of the protonated Schiff base, possesses a higher pKa ( approximately 7) compared to that of homologous Asp85 in BR (<3). This suggests that PR has a hydrogen-bonding network different from that of BR. We previously reported that a strongly hydrogen-bonded water molecule is observed only in the alkaline form of PR, where Asp97 is deprotonated (Furutani, Y., Ikeda, D., Shibata, M., and Kandori, H. (2006) Chem. Phys. 324, 705-708). This is probably correlated with the pH-dependent proton pumping activity of PR. In this work, we studied the water-containing hydrogen-bonding network in the Schiff base region of PR by means of Fourier-transform infrared (FTIR) spectroscopy at 77 K. [zeta-15N]Lys-labeling and 18O water were used for assigning the Schiff base N-D and water O-D stretching vibrations in D2O, respectively. The frequency upshift of the N-D stretch in the primary K intermediate is much smaller for PR than for BR, indicating that the Schiff base forms a hydrogen bond after retinal photoisomerization. We then measured FTIR spectra of the mutants of Asp97 (D97N and D97E) and Asp227 (D227N and D227E) to identify the amino acid interacting with the Schiff base in the K state. The PRK minus PR spectra of D97N and D97E were similar to those of the acidic and alkaline forms, respectively, of the wild type implying that the structural changes upon retinal photoisomerization are not influenced by the mutation at Asp97. In contrast, clear spectral differences were observed in D227N and D227E, including vibrational bands of the Schiff base and water molecules. It is concluded that Asp227 plays a crucial role during the photoisomerization process, though Asp97 acts as the primary counterion in the unphotolyzed state of PR.     

Coupling photoisomerization of retinal to directional transport in bacteriorhodopsin

In order to understand how isomerization of the retinal drives unidirectional transmembrane ion transport in bacteriorhodopsin, we determined the atomic structures of the BR state and M photointermediate of the E204Q mutant, to 1.7 and 1.8 A resolution, respectively. Comparison of this M, in which proton release to the extracellular surface is blocked, with the previously determined M in the D96N mutant indicates that the changes in the extracellular region are initiated by changes in the electrostatic interactions of the retinal Schiff base with Asp85 and Asp212, but those on the cytoplasmic side originate from steric conflict of the 13-methyl retinal group with Trp182 and distortion of the pi-bulge of helix G. The structural changes suggest that protonation of Asp85 initiates a cascade of atomic displacements in the extracellular region that cause release of a proton to the surface. The progressive relaxation of the strained 13-cis retinal chain with deprotonated Schiff base, in turn, initiates atomic displacements in the cytoplasmic region that cause the intercalation of a hydrogen-bonded water molecule between Thr46 and Asp96. This accounts for the lowering of the pK(a) of Asp96, which then reprotonates the Schiff base via a newly formed chain of water molecules that is extending toward the Schiff base.     

Active state of sensory rhodopsin II: structural determinants for signal transfer and proton pumping

The molecular mechanism of transmembrane signal transduction is still a pertinent question in cellular biology. Generally, a receptor can transfer an external signal via its cytoplasmic surface, as found for G-protein-coupled receptors such as rhodopsin, or via the membrane domain, such as that in sensory rhodopsin II (SRII) in complex with its transducer, HtrII. In the absence of HtrII, SRII functions as a proton pump. Here, we report on the crystal structure of the active state of uncomplexed SRII from Natronomonas pharaonis, NpSRII. The problem with a dramatic loss of diffraction quality upon loading of the active state was overcome by growing better crystals and by reducing the occupancy of the state. The conformational changes in the region comprising helices F and G are similar to those observed for the NpSRII-transducer complex but are much more pronounced. The meaning of these differences for the understanding of proton pumping and signal transduction by NpSRII is discussed.     

Structural changes due to the deprotonation of the proton release group in the M-photointermediate of bacteriorhodopsin as revealed by time-resolved FTIR spectroscopy

One of the steps in the proton pumping cycle of bacteriorhodopsin (BR) is the release of a proton from the proton-release group (PRG) on the extracellular side of the Schiff base. This proton release takes place shortly after deprotonation of the Schiff base (L-to-M transition) and results in an increase in the pKa of Asp85, which is a crucial mechanistic step for one-way proton transfer for the entire photocycle. Deprotonation of the PRG can also be brought about without photoactivation, by raising the pH of the enzyme (pKa of PRG; approximately 9). Thus, comparison of the FTIR difference spectrum for formation of the M intermediate (M minus initial unphotolyzed BR state) at pH 7 to the corresponding spectrum generated at pH 10 may reveal structural changes specifically associated with deprotonation of the PRG. Vibrational bands of BR that change upon M formation are distributed across a broad region between 2120 and 1685 cm(-1). This broad band is made up of two parts. The band above 1780 cm(-1), which is insensitive to C15-deuteration of the retinal, may be due to a proton delocalized in the PRG. The band between 1725 and 1685 cm(-1), on the lower frequency side of the broad band, is sensitive to C15-deuteration. This band may arise from transition dipole coupling of the vibrations of backbone carbonyl groups in helix G with the side chain of Tyr57 and with the C15H of the Schiff base. In M, these broad bands are abolished, and the 3657 cm(-1) band, which is due to the disruption of the hydrogen bonding of a water molecule, probably with Arg82, appears. Loss of the interaction of the backbone carbonyl groups in helix G with Tyr57 and the Schiff base, and separation of Tyr57 from Arg82, may be causes of these spectral changes, leading to the stabilization of the protonated Asp85 in M.     

Proton release and uptake of pharaonis phoborhodopsin (sensory rhodopsin II) reconstituted into phospholipids

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.     

Light-induced structural changes occur in the transmembrane helices of the Natronobacterium pharaonis HtrII transducer.

The Natronobacterium pharaonis HtrII (NpHtrII) transducer interacts with its cognate photoactive sensory rhodopsin receptor, NpSRII, to mediate phototaxis responses. NpHtrII is predicted to have two transmembrane helices and a large cytoplasmic domain and to form a homodimer. Single cysteines were substituted into an engineered cysteine-less NpHtrII at 38 positions in its transmembrane domain. Oxidative disulfide cross-linking efficiencies of the monocysteine mutants were measured with or without photoactivation of NpSRII. The rapid cross-linking rates at several positions support that NpHtrII is a dimer when functionally expressed in the Halobacterium salinarum membrane. Thirteen positions in the second transmembrane segment (TM2) exhibited significant light-induced increases in cross-linking efficiency, and they define a single face traversing the length of the segment when modeled as an alpha-helix. Four positions in this helix showing light-induced decreases in efficiency are clustered on the cytoplasmic side of the protein. One of the monocysteine mutants, G83C, showed loss of phototaxis responses, and analysis of double mutants showed that the G83C mutation alters the dark structure of the TM2-TM2' region of NpHtrII. In summary, the results reveal conformationally active regions in the second transmembrane segment of NpHtrII and a face along the length of TM2 that becomes more available for TM2-TM2' cross-linking upon receptor photoactivation. The data also establish that one residue in TM2, Gly83, is critical for maintaining the proper conformation of NpHtrII for signal relay from the photoactivated receptor to the kinase-binding region of the transducer.     

Interaction of internal water molecules with the schiff base in the L intermediate of the bacteriorhodopsin photocycle

In the photocycle of bacteriorhodopsin (BR), the first proton movement, from the Schiff base to Asp85, occurs after the formation of the L intermediate. In L, the C [double bond] N bond of the Schiff base is strained, and the nitrogen interacts strongly with its counterion. The present study seeks to detect the interaction of internal water molecules with the Schiff base in L using difference FTIR spectroscopy at 170 K. The coupled modes of the hydrogen-out-of plane bending vibrations (HOOPs) of the N-H and C(15)-H of the protonated Schiff base are detected as a broad band centered at 911 cm(-1) for BR. A set of bands at 1073, 1064, and 1056 cm(-1) for L is shown to arise from the coupling of the HOOP with the overtones of interacting water O-H vibrations. Interaction with water was shown by the decreased intensity of the HOOPs of L in H(2)(18)O and by the influence of mutants that have been shown to perturb specific internal water molecules in BR. In contrast, the HOOP band of initial BR was not affected by these mutations. In D85N, the coupled HOOP of BR is depleted, while the coupled HOOPs of L are shifted. The results indicate that the Schiff base interacts with water in the L state but in a different manner than in the BR state. Moreover, the effects of mutations suggest that cytoplasmic water close to Thr46 (Wat46) either interacts stronger with the Schiff base in L or that it is important in stabilizing another water that does.     

Cytoplasmic shuttling of protons in anabaena sensory rhodopsin: implications for signaling mechanism

It was found recently that Anabaena sensory rhodopsin (ASR), which possibly serves as a photoreceptor for chromatic adaptation, interacts with a soluble cytoplasmic transducer. The X-ray structure of the transducer-free protein revealed an extensive hydrogen-bonded network of amino acid residues and water molecules in the cytoplasmic half of ASR, in high contrast to its haloarchaeal counterparts. Using time-resolved spectroscopy of the wild-type and mutant ASR in the visible and infrared ranges, we tried to determine whether this hydrogen-bonded network is used to translocate protons and whether those proton transfers are important for interaction with the transducer. We found that the retinal Schiff base deprotonation, which occurs in the M intermediate of the photocycle of all-trans-ASR, results in protonation of Asp217 on the cytoplasmic side of the protein. The deprotonation of the Schiff base induces a conformational change of ASR observed through the perturbation of associated lipids. We suggest that the cytoplasmic shuttling of protons in the photocycle of all-trans-ASR and the ensuing conformational changes might activate the transducer. Consequently, the M intermediate may be the signaling state of ASR.