A transporter converted into a sensor, a phototaxis signaling mutant of bacteriorhodopsin at 3.0 Å.

Bacteriorhodopsin (BR) and sensory rhodopsin II (SRII), homologous photoactive proteins in haloarchaea, have different molecular functions. BR is a light-driven proton pump, whereas SRII is a phototaxis receptor that transmits a light-induced conformational change to its transducer HtrII. Despite these distinctly different functions, a single residue substitution, Ala215 to Thr215 in the BR retinal-binding pocket, enables its photochemical reactions to transmit signals to HtrII and mediate phototaxis. We pursued a crystal structure of the signaling BR mutant (BR_A215T) to determine the structural changes caused by the A215T mutation and to assess what new photochemistry is likely to be introduced into the BR photoactive site. We crystallized BR_A215T from bicelles and solved its structure to 3.0 Å resolution to enable an atomic-level comparison. The analysis was complemented by molecular dynamics simulation of BR mutated in silico. Three main conclusions regarding the roles of photoactive site residues in signaling emerge from the comparison of BR_A215T, BR, and SRII structures: (i) the Thr215 residue in signaling BR is positioned nearly identically with respect to the retinal chromophore as in SRII, consistent with its role in producing a steric conflict with the retinal C14 group during photoisomerization, proposed earlier to be essential for SRII signaling from vibrational spectroscopy and motility measurements; (ii) Tyr174-Thr204 hydrogen bonding, critical in SRII signaling and mimicked in signaling BR, is likely auxiliary, for example, to maintain Thr204 in the proper position for the steric trigger to occur; and (iii) the primary role of Arg72 in SRII is spectral tuning and not signaling.

Diversity of Chlamydomonas channelrhodopsins.

Channelrhodopsins act as photoreceptors for control of motility behavior in flagellates and are widely used as genetically targeted tools to optically manipulate the membrane potential of specific cell populations ("optogenetics"). The first two channelrhodopsins were obtained from the model organism Chlamydomonas reinhardtii (CrChR1 and CrChR2). By homology cloning we identified three new channelrhodopsin sequences from the same genus, CaChR1, CyChR1 and CraChR2, from C. augustae, C. yellowstonensis and C. raudensis, respectively. CaChR1 and CyChR1 were functionally expressed in HEK293 cells, where they acted as light-gated ion channels similar to CrChR1. However, both, which are similar to each other, differed from CrChR1 in current kinetics, inactivation, light intensity dependence, spectral sensitivity and dependence on the external pH. These results show that extensive channelrhodopsin diversity exists even within the same genus, Chlamydomonas. The maximal spectral sensitivity of CaChR1 was at 520 nm at pH 7.4, about 40 nm redshifted as compared to that of CrChR1 under the same conditions. CaChR1 was successfully expressed in Pichia pastoris and exhibited an absorption spectrum identical to the action spectrum of CaChR1-generated photocurrents. The redshifted spectra and the lack of fast inactivation in CaChR1- and CyChR1-generated currents are features desirable for optogenetics applications.

New channelrhodopsin with a red-shifted spectrum and rapid kinetics from Mesostigma viride.

Light control of motility behavior (phototaxis and photophobic responses) in green flagellate algae is mediated by sensory rhodopsins homologous to phototaxis receptors and light-driven ion transporters in prokaryotic organisms. In the phototaxis process, excitation of the algal sensory rhodopsins leads to generation of transmembrane photoreceptor currents. When expressed in animal cells, the algal phototaxis receptors function as light-gated cation channels, which has earned them the name "channelrhodopsins." Channelrhodopsins have become useful molecular tools for light control of cellular activity. Only four channelrhodopsins, identified in Chlamydomonas reinhardtii and Volvox carteri, have been reported so far. By screening light-induced currents among algal species, we identified that the phylogenetically distant flagellate Mesostigma viride showed photoelectrical responses in vivo with properties suggesting a channelrhodopsin especially promising for optogenetic use. We cloned an M. viride channelrhodopsin, MChR1, and studied its channel activity upon heterologous expression. Action spectra in HEK293 cells match those of the photocurrents observed in M. viride cells. Comparison of the more divergent MChR1 sequence to the previously studied phylogenetically clustered homologs and study of several MChR1 mutants refine our understanding of the sequence determinants of channelrhodopsin function. We found that MChR1 has the most red-shifted and pH-independent spectral sensitivity so far reported, matches or surpasses known channelrhodopsins' channel kinetics features, and undergoes minimal inactivation upon sustained illumination. This combination of properties makes MChR1 a promising candidate for optogenetic applications.

Active water in protein-protein communication within the membrane: the case of SRII-HtrII signal relay.

We detect internal water molecules in a membrane-embedded receptor-transducer complex and demonstrate water structure changes during formation of the signaling state. Time-resolved FTIR spectroscopy reveals stimulus-induced repositioning of one or more structurally active water molecules to a significantly more hydrophobic environment in the signaling state of the sensory rhodopsin II (SRII)-transducer (HtrII) complex. These waters, distinct from bound water molecules within the SRII receptor, appear to be in the middle of the transmembrane interface region near the Tyr199(SRII)-Asn74(HtrII) hydrogen bond. We conclude that water potentially plays an important role in the SRII --> HtrII signal transfer mechanism in the membrane's hydrophobic core.

His-75 in proteorhodopsin, a novel component in light-driven proton translocation by primary pumps.

Proteorhodopsins (PRs), photoactive retinylidene membrane proteins ubiquitous in marine eubacteria, exhibit light-driven proton transport activity similar to that of the well studied bacteriorhodopsin from halophilic archaea. However, unlike bacteriorhodopsin, PRs have a single highly conserved histidine located near the photoactive site of the protein. Time-resolved Fourier transform IR difference spectroscopy combined with visible absorption spectroscopy, isotope labeling, and electrical measurements of light-induced charge movements reveal participation of His-75 in the proton translocation mechanism of PR. Substitution of His-75 with Ala or Glu perturbed the structure of the photoactive site and resulted in significantly shifted visible absorption spectra. In contrast, His-75 substitution with a positively charged Arg did not shift the visible absorption spectrum of PR. The mutation to Arg also blocks the light-induced proton transfer from the Schiff base to its counterion Asp-97 during the photocycle and the acid-induced protonation of Asp-97 in the dark state of the protein. Isotope labeling of histidine revealed that His-75 undergoes deprotonation during the photocycle in the proton-pumping (high pH) form of PR, a reaction further supported by results from H75E. Finally, all His-75 mutations greatly affect charge movements within the PR and shift its pH dependence to acidic values. A model of the proteorhodopsin proton transport process is proposed as follows: (i) in the dark state His-75 is positively charged (protonated) over a wide pH range and interacts directly with the Schiff base counterion Asp-97; and (ii) photoisomerization-induced transfer of the Schiff base proton to the Asp-97 counterion disrupts its interaction with His-75 and triggers a histidine deprotonation.

Different structural changes occur in blue- and green-proteorhodopsins during the primary photoreaction.

We examine the structural changes during the primary photoreaction in blue-absorbing proteorhodopsin (BPR), a light-driven retinylidene proton pump, using low-temperature FTIR difference spectroscopy. Comparison of the light-induced BPR difference spectrum recorded at 80 K to that of green-absorbing proteorhodopsin (GPR) reveals that there are several differences in the BPR and GPR primary photoreactions despite the similar structure of the retinal chromophore and all-trans --> 13-cis isomerization. Strong bands near 1700 cm(-1) assigned previously to a change in hydrogen bonding of Asn230 in GPR are still present in BPR. However, additional bands in the same region are assigned on the basis of site-directed mutagenesis to changes occurring in Gln105. In the amide II region, bands are assigned on the basis of total (15)N labeling to structural changes of the protein backbone, although no such bands were previously observed for GPR. A band at 3642 cm(-1) in BPR, assigned to the OH stretching mode of a water molecule on the basis of H2(18)O substitution, appears at a different frequency than a band at 3626 cm(-1) previously assigned to a water molecule in GPR. However, the substitution of Gln105 for Leu105 in BPR leads to the appearance of both bands at 3642 and 3626 cm(-1), indicating the waters assigned in BPR and GPR exist in separate distinct locations and can coexist in the GPR-like Q105L mutant of BPR. These results indicate that there exist significant differences in the conformational changes occurring in these two types proteorhodopsin during the initial photoreaction despite their similar chromophore structures, which might reflect a different arrangement of water in the active site as well as substitution of a hydrophilic for hydrophobic residue at residue 105.

Raman spectroscopy reveals direct chromophore interactions in the Leu/Gln105 spectral tuning switch of proteorhodopsins.

Proteorhodopsins are an extensive family of photoactive membrane proteins found in proteobacteria distributed throughout the world's oceans which are often classified as green- or blue-absorbing (GPR and BPR, respectively) on the basis of their visible absorption maxima. GPR and BPR have significantly different properties including photocycle lifetimes and wavelength dependence on pH. Previous studies revealed that these different properties are correlated with a single residue, Leu105 in GPR and Gln105 in BPR, although the molecular basis for the different properties of GPR and BPR has not yet been elucidated. We have studied the unexcited states of GPR and BPR using resonance Raman spectroscopy which enhances almost exclusively chromophore vibrations. We find that both spectra are remarkably similar, indicating that the retinylidene structure of GPR and BPR are almost identical. However, the frequency of a band assigned to the retinal C13-methyl-rock vibration is shifted from 1006 cm (-1) in GPR to 1012 cm (-1) in BPR. A similar shift is observed in the GPR mutant L105Q indicating Leu and Gln residues interact differently with the retinal C13-methyl group. The environment of the Schiff base of GPR and BPR differ as indicated by differences in the H/D induced down-shift of the Schiff base vibration. Residues located in transmembrane helices (D-G) do not contribute to the observed differences in the protein-chromophore interaction between BPR and GPR based on the Raman spectra of chimeras. These results support a model whereby the substitution of the hydrophilic Gln105 in BPR with the smaller hydrophobic Leu105 in GPR directly alters the environment of both the retinal C13 group and the Schiff base.

Protonation state of Glu142 differs in the green- and blue-absorbing variants of proteorhodopsin.

Proteorhodopsins are a recently discovered class of microbial rhodopsins, ubiquitous in marine bacteria. Over 4000 variants have thus far been discovered, distributed throughout the oceans of the world. Most variants fall into one of two major groups, green- or blue-absorbing proteorhodopsin (GPR and BPR, respectively), on the basis of both the visible absorption maxima (530 versus 490 nm) and photocycle kinetics ( approximately 20 versus approximately 200 ms). For a well-studied pair, these differences appear to be largely determined by the identity of a single residue at position 105 (leucine/GPR and glutamine/BPR). We find using a combination of visible and infrared spectroscopy that a second difference is the protonation state of a glutamic acid residue located at position 142 on the extracellular side of helix D. In BPR, Glu142 (the GPR numbering system is used) is deprotonated and can act as an alternate proton acceptor, thus explaining the earlier observations that neutralization of the Schiff base counterion, Asp97, does not block the formation of the M intermediate. In contrast, Glu142 in GPR is protonated and cannot act in this state as an alternate proton acceptor for the Schiff base. On the basis of these findings, a mechanism is proposed for proton pumping in BPR. Because the pKa of Glu142 is near the pH of its native marine environment, changes in pH may act to modulate its function in the cell.

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.

Subpicosecond protein backbone changes detected during the green-absorbing proteorhodopsin primary photoreaction.

Recent studies demonstrate that photoactive proteins can react within several picoseconds to photon absorption by their chromophores. Faster subpicosecond protein responses have been suggested to occur in rhodopsin-like proteins where retinal photoisomerization may impulsively drive structural changes in nearby protein groups. Here, we test this possibility by investigating the earliest protein structural changes occurring in proteorhodopsin (PR) using ultrafast transient infrared (TIR) spectroscopy with approximately 200 fs time resolution combined with nonperturbing isotope labeling. PR is a recently discovered microbial rhodopsin similar to bacteriorhodopsin (BR) found in marine proteobacteria and functions as a proton pump. Vibrational bands in the retinal fingerprint (1175-1215 cm(-1)) and ethylenic stretching (1500-1570 cm(-1)) regions characteristic of all-trans to 13-cis chromophore isomerization and formation of a red-shifted photointermediate appear with a 500-700 fs time constant after photoexcitation. Bands characteristic of partial return to the ground state evolve with a 2.0-3.5 ps time constant. In addition, a negative band appears at 1548 cm(-1) with a time constant of 500-700 fs, which on the basis of total-15N and retinal C15D (retinal with a deuterium on carbon 15) isotope labeling is assigned to an amide II peptide backbone mode that shifts to near 1538 cm(-1) concomitantly with chromophore isomerization. Our results demonstrate that one or more peptide backbone groups in PR respond with a time constant of 500-700 fs, almost coincident with the light-driven retinylidene chromophore isomerization. The protein changes we observe on a subpicosecond time scale may be involved in storage of the absorbed photon energy subsequently utilized for proton transport.

Crystal structure of the Anabaena sensory rhodopsin transducer.

We present crystal structures of the Anabaena sensory rhodopsin transducer (ASRT), a soluble cytoplasmic protein that interacts with the first structurally characterized eubacterial retinylidene photoreceptor Anabaena sensory rhodopsin (ASR). Four crystal structures of ASRT from three different spacegroups were obtained, in all of which ASRT is present as a planar (C4) tetramer, consistent with our characterization of ASRT as a tetramer in solution. The ASRT tetramer is tightly packed, with large interfaces where the well-structured beta-sandwich portion of the monomers provides the bulk of the tetramer-forming interactions, and forms a flat, stable surface on one side of the tetramer (the beta-face). Only one of our four different ASRT crystals reveals a C-terminal alpha-helix in the otherwise all-beta protein, together with a large loop from each monomer on the opposite face of the tetramer (the alpha-face), which is flexible and largely disordered in the other three crystal forms. Gel-filtration chromatography demonstrated that ASRT forms stable tetramers in solution and isothermal microcalorimetry showed that the ASRT tetramer binds to ASR with a stoichiometry of one ASRT tetramer per one ASR photoreceptor with a K(d) of 8 microM in the highest affinity measurements. Possible mechanisms for the interaction of this transducer tetramer with the ASR photoreceptor via its flexible alpha-face to mediate transduction of the light signal are discussed.

Role of the cytoplasmic domain in Anabaena sensory rhodopsin photocycling: vectoriality of Schiff base deprotonation.

Light-induced electric signals in intact E. coli cells generated by heterologously expressed full-length and C-terminally truncated versions of Anabaena sensory rhodopsin (ASR) demonstrate that the charge movements within the membrane-embedded part of the molecule are stringently controlled by the cytoplasmic domain. In particular, truncation inverts the direction of proton movement during Schiff base deprotonation from outward to cytoplasmic. Truncation also alters faster charge movements that occur before Schiff base deprotonation. Asp(217) as previously shown by FTIR serves as a proton acceptor in the truncated ASR but not in the full-length version, and its mutation to Asn restores the natural outward direction of proton movement. Introduction of a potential negative charge (Ser(86) to Asp) on the cytoplasmic side favors a cytoplasmic direction of proton release from the Schiff base. In contrast, mutation of the counterion Asp(75) to Glu reverses the photocurrent to the outward direction in the truncated pigment, and in both truncated and full-length versions accelerates Schiff base deprotonation more than 10-fold. The communication between the cytoplasmic domain and the membrane-embedded photoactive site of ASR demonstrated here is likely to derive from the receptor's use of a cytoplasmic protein for signal transduction, as has been suggested previously from binding studies.

Photoactivation perturbs the membrane-embedded contacts between sensory rhodopsin II and its transducer.

The photoactivation mechanism of the sensory rho-dopsin II (SRII)-HtrII receptor-transducer complex of Natronomonas pharaonis was investigated by time-resolved Fourier transform infrared difference spectroscopy to identify structural changes associated with early events in the signal relay mechanism from the receptor to the transducer. Several prominent bands in the wild-type SRII-HtrII spectra are affected by amino acid substitutions at the receptor Tyr(199) and transducer Asn(74) residues, which form a hydrogen bond between the two proteins near the middle of the bilayer. Our results indicate disappearance of this hydrogen bond in the M and O photointermediates, the likely signaling states of the complex. This event represents one of the largest light-induced alterations in the binding contacts between the receptor and transducer. The vibrational frequency changes suggest that Asn(74) and Tyr(199) form other stronger hydrogen bonds in the M state. The light-induced disruption of the Tyr(199)-Asn(74) bond also occurs when the Schiff base counterion Asp(75) is replaced with a neutral asparagine. We compared the decrease in intensity of difference bands assigned to the Tyr(199)-Asn(74) pair and to chromophore and protein groups of the receptor at various time points during the recovery of the initial state. All difference bands exhibit similar decay kinetics indicating that reformation of the Tyr(199)-Asn(74) hydrogen bond occurs concomitantly with the decay of the M and O photointermediates. This work demonstrates that the signal relay from SRII to HtrII involves early structural alterations in the deeply membrane-embedded domain of the complex and provides a spectroscopic signal useful for correlation with the downstream events in signal transduction.

Structural changes in the photoactive site of proteorhodopsin during the primary photoreaction.

Proteorhodopsin (PR), found in marine gamma-proteobacteria, is a newly discovered light-driven proton pump similar to bacteriorhodopsin (BR). Because of the widespread distribution of proteobacteria in the worldwide oceanic waters, this pigment may contribute significantly to the global solar energy input in the biosphere. We examined structural changes that occur during the primary photoreaction (PR --> K) of wild-type pigment and two mutants using low-temperature FTIR difference spectroscopy. Several vibrations detected in the 3500-3700 cm(-1) region are assigned on the basis of H(2)O --> H(2)(18)O exchange to the perturbation of one or more internal water molecules. Substitution of the negatively charged Schiff base counterion, Asp97, with the neutral asparagine caused a downshift of the ethylenic (C=C) and Schiff base (C=N) stretching modes, in agreement with the 27 nm red shift of the visible lambda(max). However, this replacement did not alter the normal all-trans to 13-cis isomerization of the chromophore or the environment of the detected water molecule(s). In contrast, substitution of Asn230, which is in a position to interact with the Schiff base, with Ala induces a 5 nm red shift of the visible lambda(max) and alters the PR chromophore structure, its isomerization to K, and the environment of the detected internal water molecules. The combination of FTIR and site-directed mutagenesis establishes that both Asp97 and Asn230 are perturbed during the primary phototransition. The environment of Asn230 is further altered during the thermal decay of K. These results suggest that significant differences exist in the conformational changes which occur in the photoactive sites of proteorhodopsin and bacteriorhodopsin during the primary photoreaction.

The cytoplasmic membrane-proximal domain of the HtrII transducer interacts with the E-F loop of photoactivated Natronomonas pharaonis sensory rhodopsin II.

The structures of the cytoplasmic loops of the phototaxis receptor sensory rhodopsin II (SRII) and the membrane-proximal cytoplasmic domain of its bound transducer HtrII were examined in the dark and in the light-activated state by fluorescent probes and cysteine cross-linking. Light decreased the accessibility of E-F loop position 154 in the SRII-HtrII complex, but not in free SRII, consistent with HtrII proximity, which was confirmed by tryptophans placed within a 5-residue region identified in the HtrII membrane-proximal domain that exhibited Forster resonance energy transfer to a fluorescent probe at position 154 in SRII. The Forster resonance energy transfer was eliminated in the signaling deficient HtrII mutant G83F without loss of affinity for SRII. Finally, the presence of SRII and HtrII reciprocally inhibit homodimer disulfide cross-linking reactions in their membrane-proximal domains, showing that each interferes with the others self-interaction in this region. The results demonstrate close proximity between SRII-HtrII in the membrane-proximal domain, and in addition, light stimulation of the SRII inhibition of HtrII cross-linking was observed, indicating that the contact is enhanced in the photoactivated complex. A mechanism is proposed in which photoactivation alters the SRII-HtrII interaction in the membrane-proximal region during the signal relay process.

Characterization of RS29, a blue-green proteorhodopsin variant from the Red Sea.

Using structural modeling comparisons and mutagenesis, amino acid residue 105 was found to function as a spectral tuning switch in marine proteorhodopsins (PR). Changes at this position account for most of the spectral difference between blue-absorbing PRs (B-PRs), and green-absorbing PRs (G-PRs). Here we analyzed a Red Sea variant (RS29) from a new family of PRs that is composed of G-PR type variants that possess glutamine instead of leucine at position 105 like in B-PRs. The absorption spectrum as well as photocycle of RS29 variant were measured and compared to point-mutated 'position 105' PRs. Unexpectedly, the absorption maximum of RS29 was 515 nm, a smaller blue shift compared to the 498 nm maximum of G-PR_L105Q. We found that two additional residues at positions 65 and 70 each contribute a small red shift to the absorption spectrum of G-PR and therefore appear to account for the intermediate absorption maximum of RS29 by their opposing influences on the spectrum. Our results show that in addition to the retinal pocket position 105 determinant, other residues predicted to be outside the retinal pocket fine-tune the absorption spectra of marine PRs. The RS29 photochemical reaction cycle was found to be 2 orders of magnitude slower than that of G-PR with a t(1/2) of >600 ms. This result raises the possibility of regulatory (i.e. sensory) rather than energy harvesting functions for some members of the PR family.

Spectroscopic and photochemical characterization of a deep ocean proteorhodopsin.

A second group of proteorhodopsin-encoding genes (blue-absorbing proteorhodopsin, BPR) differing by 20-30% in predicted primary structure from the first-discovered green-absorbing (GPR) group has been detected in picoplankton from Hawaiian deep sea water. Here we compare BPR and GPR absorption spectra, photochemical reactions, and proton transport activity. The photochemical reaction cycle of Hawaiian deep ocean BPR in cells is 10-fold slower than that of GPR with very low accumulation of a deprotonated Schiff base intermediate in cells and exhibits mechanistic differences, some of which are due to its glutamine residue rather than leucine at position 105. In contrast to GPR and other characterized microbial rhodopsins, spectral titrations of BPR indicate that a second titratable group, in addition to the retinylidene Schiff base counterion Asp-97, modulates the absorption spectrum near neutral pH. Mutant analysis confirms that Asp-97 and Glu-108 are proton acceptor and proton donor, respectively, in retinylidene Schiff base proton transfer reactions during the BPR photocycle as previously shown for GPR, but BPR contains an alternative acceptor evident in its D97N mutant, possibly the same as the second titratable group modulating the absorption spectrum. BPR, similar to GPR, carries out outward light-driven proton transport in Escherichia coli vesicles but with a reduced translocation rate attributable to its slower photocycle. In energized E. coli cells at physiological pH, the net effect of BPR photocycling is to generate proton currents dominated by a triggered proton influx, rather than efflux as observed with GPR-containing cells. Reversal of the proton current with the K+-ionophore valinomycin supports that the influx is because of voltage-gated channels in the E. coli cell membrane. These observations demonstrate diversity in photochemistry and mechanism among proteorhodopsins. Calculations of photon fluence rates at different ocean depths show that the difference in photocycle rates between GPR and BPR as well as their different absorption maxima may be explained as an adaptation to the different light intensities available in their respective marine environments. Finally, the results raise the possibility of regulatory (i.e. sensory) rather than energy harvesting functions of some members of the proteorhodopsin family.

Conformational changes detected in a sensory rhodopsin II-transducer complex.

Sensory rhodopsins (SRs) are light receptors that belong to the growing family of microbial rhodopsins. SRs have now been found in all three major domains of life including archaea, bacteria, and eukaryotes. One of the most extensively studied sensory rhodopsins is SRII, which controls a blue light avoidance motility response in the halophilic archaeon Natronobacterium pharaonis. This seven-helix integral membrane protein forms a tight intermolecular complex with its cognate transducer protein, HtrII. In this work, the structural changes occurring in a fusion complex consisting of SRII and the two transmembrane helices (TM1 and TM2) of HtrII were investigated by time-resolved Fourier transform infrared difference spectroscopy. Although most of the structural changes observed in SRII are conserved in the fusion complex, several distinct changes are found. A reduction in the intensity of a prominent amide I band observed for SRII indicates that its structural changes are altered in the fusion complex, possibly because of the close interaction of TM2 with the F helix, which interferes with the F helix outward tilt. Deprotonation of at least one Asp/Glu residue is detected in the transducer-free receptor with a pKa near 7 that is abolished or altered in the fusion complex. Changes are also detected in spectral regions characteristic of Asn and Tyr vibrations. At high hydration levels, transducer-fusion interactions lead to a stabilization of an M-like intermediate that most likely corresponds to an active signaling form of the transducer. These findings are discussed in the context of a recently elucidated x-ray structure of the fusion complex.

Diversification and spectral tuning in marine proteorhodopsins.

Proteorhodopsins, ubiquitous retinylidene photoactive proton pumps, were recently discovered in the cosmopolitan uncultured SAR86 bacterial group in oceanic surface waters. Two related proteorhodopsin families were found that absorb light with different absorption maxima, 525 nm (green) and 490 nm (blue), and their distribution was shown to be stratified with depth. Using structural modeling comparisons and mutagenesis, we report here on a single amino acid residue at position 105 that functions as a spectral tuning switch and accounts for most of the spectral difference between the two pigment families. Furthermore, looking at natural environments, we found novel proteorhodopsin gene clusters spanning the range of 540-505 nm and containing changes in the same identified key switch residue leading to changes in their absorption maxima. The results suggest a simultaneous diversification of green proteorhodopsin and the new key switch variant pigments. Our observations demonstrate that this single-residue switch mechanism is the major determinant of proteorhodopsin wavelength regulation in natural marine environments.

A Fourier transform infrared study of Neurospora rhodopsin: similarities with archaeal rhodopsins.

The NOP-1 gene from the eukaryote Neurospora crassa, a filamentous fungus, has recently been shown to encode an archaeal rhodopsin-like protein NOP-1. To explore the functional mechanism of NOP-1 and its possible similarities to archaeal and visual rhodopsins, static and time-resolved Fourier transform infrared difference spectra were measured from wild-type NOP-1 and from a mutant containing an Asp-->Glu substitution in the Schiff base (SB) counterion, Asp131 (D131E). Several conclusions could be drawn about the molecular mechanism of NOP-1: (1) the NOP-1 retinylidene chromophore undergoes an all-trans to 13-cis isomerization, which is typical of archaeal rhodopsins, and closely resembles structural changes of the chromophore in sensory rhodopsin II; (2) the NOP-1 SB counterion, Asp131, has a very similar environment and behavior compared with the SB counterions in bacteriorhodopsin (BR) and sensory rhodopsin II; (3) the O-H stretching of a structurally active water molecule(s) in NOP-1 is similar to water detected in BR and is most likely located near the SB and SB counterion in these proteins; and (4) one or more cysteine residues undergo structural changes during the NOP-1 photocycle. Overall, these results indicate that many features of the active sites of the archaeal rhodopsins are conserved in NOP-1, despite its eukaryotic origin.