An archaeal photosignal-transducing module mediates phototaxis in Escherichia coli.

Halophilic archaea, such as Halobacterium salinarum and Natronobacterium pharaonis, alter their swimming behavior by phototaxis responses to changes in light intensity and color using visual pigment-like sensory rhodopsins (SRs). In N. pharaonis, SRII (NpSRII) mediates photorepellent responses through its transducer protein, NpHtrII. Here we report the expression of fusions of NpSRII and NpHtrII and fusion hybrids with eubacterial cytoplasmic domains and analyze their function in vivo in haloarchaea and in eubacteria. A fusion in which the C terminus of NpSRII is connected by a short flexible linker to NpHtrII is active in phototaxis signaling for H. salinarum, showing that the fusion does not inhibit functional receptor-transducer interactions. We replaced the cytoplasmic portions of this fusion protein with the cytoplasmic domains of Tar and Tsr, chemotaxis transducers from enteric eubacteria. Purification of the fusion protein from H. salinarum and Tar fusion chimera from Escherichia coli membranes shows that the proteins are not cleaved and exhibit absorption spectra characteristic of wild-type membranes. Their photochemical reaction cycles in H. salinarum and E. coli membranes, respectively, are similar to those of native NpSRII in N. pharaonis. These fusion chimeras mediate retinal-dependent phototaxis responses by Escherichia coli, establishing that the nine-helix membrane portion of the receptor-transducer complex is a modular functional unit able to signal in heterologous membranes. This result confirms a current model for SR-Htr signal transduction in which the Htr transducers are proposed to interact physically and functionally with their cognate sensory rhodopsins via helix-helix contacts between their transmembrane segments.

Photochemical reaction cycle and proton transfers in Neurospora rhodopsin.

It was recently found that NOP-1, a membrane protein of Neurospora crassa, shows homology to haloarchaeal rhodopsins and binds retinal after heterologous expression in Pichia pastoris. We report on spectroscopic properties of the Neurospora rhodopsin (NR). The photocycle was studied with flash photolysis and time-resolved Fourier-transform infrared spectroscopy in the pH range 5-8. Proton release and uptake during the photocycle were monitored with the pH-sensitive dye, pyranine. Kinetic and spectral analysis revealed six distinct states in the NR photocycle, and we describe their spectral properties and pH-dependent kinetics in the visible and infrared ranges. The phenotypes of the mutant NR proteins, D131E and E142Q, in which the homologues of the key carboxylic acids of the light-driven proton pump bacteriorhodopsin, Asp-85 and Asp-96, were replaced, show that Glu-142 is not involved in reprotonation of the Schiff base but Asp-131 may be. This implies that, if the NR photocycle is associated with proton transport, it has a low efficiency, similar to that of haloarchaeal sensory rhodopsin II. Fourier-transform Raman spectroscopy revealed unexpected differences between NR and bacteriorhodopsin in the configuration of the retinal chromophore, which may contribute to the less effective reprotonation switch of NR.

Proteorhodopsin phototrophy in the ocean.

Proteorhodopsin, a retinal-containing integral membrane protein that functions as a light-driven proton pump, was discovered in the genome of an uncultivated marine bacterium; however, the prevalence, expression and genetic variability of this protein in native marine microbial populations remain unknown. Here we report that photoactive proteorhodopsin is present in oceanic surface waters. We also provide evidence of an extensive family of globally distributed proteorhodopsin variants. The protein pigments comprising this rhodopsin family seem to be spectrally tuned to different habitats--absorbing light at different wavelengths in accordance with light available in the environment. Together, our data suggest that proteorhodopsin-based phototrophy is a globally significant oceanic microbial process.

Crystal structure of sensory rhodopsin II at 2.4 angstroms: insights into color tuning and transducer interaction.

We report an atomic-resolution structure for a sensory member of the microbial rhodopsin family, the phototaxis receptor sensory rhodopsin II (NpSRII), which mediates blue-light avoidance by the haloarchaeon Natronobacterium pharaonis. The 2.4 angstrom structure reveals features responsible for the 70- to 80-nanometer blue shift of its absorption maximum relative to those of haloarchaeal transport rhodopsins, as well as structural differences due to its sensory, as opposed to transport, function. Multiple factors appear to account for the spectral tuning difference with respect to bacteriorhodopsin: (i) repositioning of the guanidinium group of arginine 72, a residue that interacts with the counterion to the retinylidene protonated Schiff base; (ii) rearrangement of the protein near the retinal ring; and (iii) changes in tilt and slant of the retinal polyene chain. Inspection of the surface topography reveals an exposed polar residue, tyrosine 199, not present in bacteriorhodopsin, in the middle of the membrane bilayer. We propose that this residue interacts with the adjacent helices of the cognate NpSRII transducer NpHtrII.

Electron crystallographic analysis of two-dimensional crystals of sensory rhodopsin II: a 6.9 A projection structure.

Sensory rhodopsins, phototaxis receptors in Haloarchaea, were purified and reconstituted into halobacterial lipids to form photoactive two-dimensional crystals. Images of vitreous ice-embedded, flattened, tubular crystals of sensory rhodopsin II (SRII) of Natronobacterium pharaonis were recorded using a field emission gun electron cryo-microscope. Fourier components for the SRII structure were determined either from the separated image transforms from single layers that formed each side of flattened tubes, or by a deconvolution procedure when two layers were stacked in register so that they generated a single crystal lattice by superposition. Most micrographs showed significant diffraction to 6.9 A after computer processing, and the results provide the first intermediate- resolution information obtained for an archaeal sensory rhodopsin. The projection structure of SRII indicates that the helix positions match the seven-helix arrangement of the archaeal transport rhodopsins rather than that of the eukaryotic visual pigments. The structural similarity of SRII to the transport rhodopsins supports models in which the transport and signalling mechanisms of archaeal rhodopsins derive from the same retinal-driven changes in protein conformation.

VirB7 lipoprotein is exocellular and associates with the Agrobacterium tumefaciens T pilus.

Agrobacterium tumefaciens transfers oncogenic T-DNA and effector proteins to plant cells via a type IV secretion pathway. This transfer system, assembled from the products of the virB operon, is thought to consist of a transenvelope mating channel and the T pilus. When screened for the presence of VirB and VirE proteins, material sheared from the cell surface of octopine strain A348 was seen to possess detectable levels of VirB2 pilin, VirB5, and the VirB7 outer membrane lipoprotein. Material sheared from the cell surface of most virB gene deletion mutants also possessed VirB7, but not VirB2 or VirB5. During purification of the T pilus from wild-type cells, VirB2, VirB5, and VirB7 cofractionated through successive steps of gel filtration chromatography and sucrose density gradient centrifugation. A complex containing VirB2 and VirB7 was precipitated from a gel filtration fraction enriched for T pilus with both anti-VirB2 and anti-VirB7 antiserum. Both the exocellular and cellular forms of VirB7 migrated as disulfide-cross-linked dimers and monomers when samples were electrophoresed under nonreducing conditions. A mutant synthesizing VirB7 with a Ser substitution of the lipid-modified Cys15 residue failed to elaborate the T pilus, whereas a mutant synthesizing VirB7 with a Ser substitution for the disulfide-reactive Cys24 residue produced very low levels of T pilus. Together, these findings establish that the VirB7 lipoprotein localizes exocellularly, it associates with the T pilus, and both VirB7 lipid modification and disulfide cross-linking are important for T-pilus assembly. T-pilus-associated VirB2 migrated in nonreducing gels as a monomer and a disulfide-cross-linked homodimer, whereas cellular VirB2 migrated as a monomer. A strain synthesizing a VirB2 mutant with a Ser substitution for the reactive Cys64 residue elaborated T pilus but exhibited an attenuated virulence phenotype. Dithiothreitol-treated T pilus composed of native VirB2 pilin and untreated T pilus composed of the VirB2C64S mutant pilin distributed in sucrose gradients more predominantly in regions of lower sucrose density than untreated, native T pili. These findings indicate that intermolecular cross-linking of pilin monomers is not required for T-pilus production, but cross-linking does contribute to T-pilus stabilization.

Retinylidene proteins: structures and functions from archaea to humans.

Retinylidene proteins, containing seven membrane-embedded alpha-helices that form an internal pocket in which the chromophore retinal is bound, are ubiquitous in photoreceptor cells in eyes throughout the animal kingdom. They are also present in a diverse range of other organisms and locations, such as archaeal prokaryotes, unicellular eukaryotic microbes, the dermal tissue of frogs, the pineal glands of lizards and birds, the hypothalamus of toads, and the human brain. Their functions include light-driven ion transport and phototaxis signaling in microorganisms, and retinal isomerization and various types of photosignal transduction in higher animals. The aims of this review are to examine this group of photoactive proteins as a whole, to summarize our current understanding of structure/function relationships in the best-studied examples, and to report recent new developments.

Bacterial rhodopsin: evidence for a new type of phototrophy in the sea.

Extremely halophilic archaea contain retinal-binding integral membrane proteins called bacteriorhodopsins that function as light-driven proton pumps. So far, bacteriorhodopsins capable of generating a chemiosmotic membrane potential in response to light have been demonstrated only in halophilic archaea. We describe here a type of rhodopsin derived from bacteria that was discovered through genomic analyses of naturally occuring marine bacterioplankton. The bacterial rhodopsin was encoded in the genome of an uncultivated gamma-proteobacterium and shared highest amino acid sequence similarity with archaeal rhodopsins. The protein was functionally expressed in Escherichia coli and bound retinal to form an active, light-driven proton pump. The new rhodopsin exhibited a photochemical reaction cycle with intermediates and kinetics characteristic of archaeal proton-pumping rhodopsins. Our results demonstrate that archaeal-like rhodopsins are broadly distributed among different taxa, including members of the domain Bacteria. Our data also indicate that a previously unsuspected mode of bacterially mediated light-driven energy generation may commonly occur in oceanic surface waters worldwide.

FTIR analysis of the SII540 intermediate of sensory rhodopsin II: Asp73 is the Schiff base proton acceptor.

Sensory rhodopsin II (SRII), a repellent phototaxis receptor found in Halobacterium salinarum, has several homologous residues which have been found to be important for the proper functioning of bacteriorhodopsin (BR), a light-driven proton pump. These include Asp73, which in the case of bacteriorhodopsin (Asp85) functions as the Schiff base counterion and proton acceptor. We analyzed the photocycles of both wild-type SRII and the mutant D73E, both reconstituted in Halobacterium salinarum lipids, using FTIR difference spectroscopy under conditions that favor accumulation of the O-like, photocycle intermediate, SII540. At both room temperature and -20 degrees C, the difference spectrum of SRII is similar to the BR-->O640 difference spectrum of BR, especially in the configurationally sensitive retinal fingerprint region. This indicates that SII540 has an all-trans chromophore similar to the O640 intermediate in BR. A positive band at 1761 cm-1 downshifts 40 cm-1 in the mutant D73E, confirming that Asp73 undergoes a protonation reaction and functions in analogy to Asp85 in BR as a Schiff base proton acceptor. Several other bands in the C=O stretching regions are identified which reflect protonation or hydrogen bonding changes of additional Asp and/or Glu residues. Intense bands in the amide I region indicate that a protein conformational change occurs in the late SRII photocycle which may be similar to the conformational changes that occur in the late BR photocycle. However, unlike BR, this conformational change does not reverse during formation of the O-like intermediate, and the peptide groups giving rise to these bands are partially accessible for hydrogen/deuterium exchange. Implications of these findings for the mechanism of SRII signal transduction are discussed.

A eukaryotic protein, NOP-1, binds retinal to form an archaeal rhodopsin-like photochemically reactive pigment.

The nop-1 gene from Neurospora crassa is predicted to encode a seven-helix protein exhibiting conservation with the rhodopsins of the archaeon Halobacterium salinarum. In the work presented here we have expressed this gene heterologously in the yeast Pichia pastoris, obtaining a relatively high yield of 2.2 mg of NOP-1 protein/L of cell culture. The expressed protein is membrane-associated and forms with all-trans retinal a visible light-absorbing pigment with a 534 nm absorption maximum and approximately 100 nm half-bandwidth typical of retinylidene protein absorption spectra. Its lambda(max) indicates a protonated Schiff base linkage of the retinal. Laser flash kinetic spectroscopy demonstrates that the retinal-reconstituted pigment undergoes a photochemical reaction cycle with a near-UV-absorbing intermediate that is similar to the M intermediates produced by transient Schiff base deprotonation of the chromophore in the photocycles of bacteriorhodopsin and sensory rhodopsins I and II. The slow photocycle (seconds) and long-lived intermediates (M and O) are most similar to those of the phototaxis receptor sensory rhodopsin II. The results demonstrate a photochemically reactive member of the archaeal rhodopsin family in a eukaryotic cell.

Transducer-binding and transducer-mutations modulate photoactive-site-deprotonation in sensory rhodopsin I.

Sensory rhodopsin I (SRI) is a seven-transmembrane helix retinylidene protein that mediates color-sensitive phototaxis responses through its bound transducer HtrI in the archaeon Halobacterium salinarum. Deprotonation of the Schiff base attachment site of the chromophore accompanies formation of the SRI signaling state, S(373). We measured the rate of laser flash-induced S(373) formation in the presence and absence of HtrI, and the effects of mutations in SRI or HtrI on the kinetics of this process. In the absence of HtrI, deprotonation occurs rapidly (halftime 10 micros) if the proton acceptor Asp76 is ionized (pK(a) = approximately 7), and only very slowly (halftime > 10 ms) when Asp76 is protonated. Transducer-binding, although it increases the pK(a) of Asp76 so that it is protonated throughout the range of pH studied, results in a first order, pH-independent rate of S(373) formation of approximately 300 micros. Therefore, the complexation of HtrI facilitates the proton-transfer reaction, increasing the rate approximately 50-fold at pH6. Arrhenius analysis shows that HtrI-binding accelerates the reaction primarily by an entropic effect, suggesting HtrI constrains the SRI molecule in the complex. Function-perturbing mutations in SRI and HtrI also alter the rate of S(373) formation and the lambda(max) of the parent state as assessed by laser flash-induced kinetic difference spectroscopy, and shifts to longer wavelength are correlated with slower deprotonation. The data indicate that HtrI affects electrostatic interactions of the protonated Schiff base and not only receives the signal from SRI but also optimizes the photochemical reaction process for SRI signaling.

Constitutive signaling by the phototaxis receptor sensory rhodopsin II from disruption of its protonated Schiff base-Asp-73 interhelical salt bridge.

Sensory rhodopsin II (SRII) is a repellent phototaxis receptor in the archaeon Halobacterium salinarum, similar to visual pigments in its seven-helix structure and linkage of retinal to the protein by a protonated Schiff base in helix G. Asp-73 in helix C is shown by spectroscopic analysis to be a counterion to the protonated Schiff base in the unphotolyzed SRII and to be the proton acceptor from the Schiff base during photoconversion to the receptor signaling state. Coexpression of the genes encoding mutated SRII with Asn substituted for Asp-73 (D73N) and the SRII transducer HtrII in H. salinarum cells results in a 3-fold higher swimming reversal frequency accompanied by demethylation of HtrII in the dark, showing that D73N SRII produces repellent signals in its unphotostimulated state. Analogous constitutive signaling has been shown to be produced by the similar neutral residue substitution of the Schiff base counterion and proton acceptor Glu-113 in human rod rhodopsin. The interpretation for both seven-helix receptors is that light activation of the wild-type protein is caused primarily by photoisomerization-induced transfer of the Schiff base proton on helix G to its primary carboxylate counterion on helix C. Therefore receptor activation by helix C-G salt-bridge disruption in the photoactive site is a general mechanism in retinylidene proteins spanning the vast evolutionary distance between archaea and humans.

Effects of substitutions D73E, D73N, D103N and V106M on signaling and pH titration of sensory rhodopsin II.

Several mutations in the repellent phototaxis receptor sensory rhodopsin II (SRII), in residues homologous to residues important in the related proton pump bacteriorhodopsin, were expressed in Pho81Wr-, a Halobacterium salinarum strain deficient in production of SRII and its transducer protein HtrII. The lack of production of SRII and HtrII is shown to be due to insertion of an ISH2 transposon into the promoter region upstream of the htrII-sopII gene pair. Near wild-type phototaxis responses are rescued in Pho81Wr- by expression of HtrII with D73E, D103N or V106M receptors. Partial responses are restored by the HtrII-D73N pair. From absorption spectroscopy of his-tag-purified receptor protein from mutants D73N and D73E we conclude that Asp73 is the primary counterion to the protonated Schiff base in SRII, like the corresponding Asp85 in bacteriorhodopsin. The absorption maximum of SRII (487 nm) is shifted to 514 nm in mutant D73N, a 1080 cm-1 shift identical to that caused by D85N in bacteriorhodopsin. Acid titration of SRII also induces the red shift with a pK of 3.0 in wild type. The absorption shift and the pK are nearly the same in V106M and D103N, but the pK is raised to 5.1 in D73E, confirming that Asp73 is the residue responsible for this spectral transition.

Deletion mapping of the sites on the HtrI transducer for sensory rhodopsin I interaction.

The phototaxis receptor sensory rhodopsin I (SRI) transmits signals through a membrane-bound transducer protein, HtrI. The genes for the receptor and transducer, sopI and htrI, respectively, are normally cotranscribed; however, previous work has established that fully functional interacting proteins are produced when htrI is expressed from the chromosome and sopI is expressed from a different promoter on a plasmid. In this report we show that in the membrane, concentrations of SRI from plasmid expression of wild-type sopI are negligible in the absence of HtrI protein in the cell. This requirement for HtrI is eliminated when sopI is extended at the 5'-end with 63 nucleotides of the bop gene, which encodes the N-terminal signal sequence of the bacteriorhodopsin protein. The signal is cleaved from the chimeric protein, and processed SRI is stable in the HtrI-free membrane. These results suggest a chaperone-like function for HtrI that facilitates membrane insertion or proper folding of the SRI protein. Six deletion constructs of HtrI were examined to localize the interaction sites for its putative chaperone function and for HtrI control of the SRI photocycle, a phenomenon described previously. The smallest HtrI fragment identified, which contained interaction sites for both SRI stability and photocycle control, consisted of the N-terminal 147 residues of the 536-residue HtrI protein. The active fragment is predicted to contain two transmembrane helices and the first approximately 20% of the cytoplasmic portion of the protein.

Asp76 is the Schiff base counterion and proton acceptor in the proton-translocating form of sensory rhodopsin I.

Both sensory rhodopsin I, a phototaxis receptor, and bacteriorhodopsin, a light-driven proton pump, have homologous residues which have been identified as critical for bacteriorhodopsin functioning. This includes Asp76, which in the case of bacteriorhodopsin (Asp85) functions as both the Schiff base counterion and the proton acceptor. Sensory rhodopsin I exists in a pH dependent equilibrium between two different forms in the absence of its transducer protein HtrI. At pH below 7, it exists primarily in a blue form (lambda max = 587 nm) which functions as a phototaxis signal transducer when complexed to HtrI, while at higher pH, it converts to a purple proton-transporting form similar to bacteriorhodopsin (lambda max = 550 nm). We report ATR-FTIR difference spectra obtained from both low- and high-pH forms of purified sensory rhodopsin I reconstituted into lipid vesicles. The low-pH species has an ethylenic C = C stretch mode at 1520 cm-1 which shifts to 1526 cm-1 in the high-pH form. No frequency shift was found for the mutant D76N, in agreement with visible absorption measurements. Weak negative/positive bands at 1763/1751 cm-1 previously assigned to a perturbation of the C = O stretch mode of Asp76 during S373 formation in the low-pH form are replaced by a single intense positive band near 1749 cm-1 in the high-pH form. These results along with the effects of H/D exchange show that Asp76 is protonated in the signal-transducing form of sensory rhodopsin I and is ionized and functions as the counterion and Schiff base proton acceptor in the proton-transporting high-pH form of sensory rhodopsin I similar to bacteriorhodopsin.

Rapid high-yield purification and liposome reconstitution of polyhistidine-tagged sensory rhodopsin I.

We have used Ni(2+)-affinity chromatography as a rapid and efficient method to purify a sensory rhodopsin I (SR-I) derivative containing six consecutive histidine residues at its C-terminus (His-tagged SR-I). The protein was expressed in Halobacterium salinarium by integrating the corresponding gene at the chromosomal bacterioopsin locus under the control of the bacterioopsin promoter. His-tagged SR-I retains native SR-I photochemical reactions in purified membranes and phototaxis signaling function in vivo. Immobilized Ni(2+)-affinity chromatography of membranes solubilized in 1% layryl maltoside provides a single-step purification of the protein to electrophoretic homogeneity (> or = 90% pure). The procedure yields 1.7 mg pure photoactive protein/liter of culture (60% efficiency). This yield combined with engineered overproduction of the protein provides at least 120-fold greater amounts than that of a previously reported multistep purification procedure, permitting structural and biochemical analysis previously not feasible. The purified protein in lauryl maltoside at pH 5.3 exhibits a visible absorption maximum at 587 nm characteristic of SR-I. Spectrometric titration reveals an alkaline-induced species at 550 nm previously observed with transducer-free SR-I in native membranes. A previously unreported structured absorption band at 400 nm, consistent with a deprotonated Schiff base, forms with the same pKa as the 550-nm species. His-tagged SR-I reconstituted into phosphatidylglycerol proteoliposomes retains properties of transducer-free SR-I in native membranes: its flash-induced absorption difference spectrum is identical, its photochemical reaction cycle kinetics show a similar pH dependence, and it forms a photoactive 550-nm species under alkaline conditions. These results indicate His-tagged SR-I reconstituted in proteoliposomes is suitable for analyzing SR-I interaction with its transducer protein in vitro.

Identification of distinct domains for signaling and receptor interaction of the sensory rhodopsin I transducer, HtrI.

The phototaxis-deficient mutant of Halobacterium salinarium, Pho81, lacks both sensory rhodopsin I (SR-I) and its putative transducer protein HtrI, according to immunoblotting and spectroscopic criteria. From restriction analysis and selected DNA sequencing, we have determined that the SR-I- HtrI- phenotype results from an insertion of a 520-bp transposable element, ISH2, into the coding region of the SR-I apoprotein gene sopI and deletion of 11 kbp upstream of ISH2 including the first 164 bp of sopI and the entire htrI gene. SR-I and HtrI expression as well as full phototaxis sensitivity are restored by transformation with a halobacterial plasmid carrying the htrI-sopI gene pair and their upstream promoter region. An internal deletion of a portion of htrI encoding the putative methylation and signaling domains of HtrI (253 residues) prevents the restoration of phototaxis, providing further evidence for the role of HtrI as a transducer for SR-I. Analysis of flash-induced photochemical reactions of SR-I over a range of pH shows that the partially deleted HtrI maintains SR-I interactions sites responsible for modulation of the SR-I photocycle.

The photochemical reactions of sensory rhodopsin I are altered by its transducer.

The sopI gene, which encodes the phototaxis receptor sensory rhodopsin I (SR-I), was expressed in Halobacterium salinarium strains with chromosomal deletions of (i) sopI only or of (ii) the region containing sopI and htrI. The htrI gene encodes a transducer protein for SR-I signals. Transformation of the sopI deletion mutant containing the htrI gene by a multicopy expression plasmid for sopI results in normal physiological and photochemical properties of SR-I. Transformation by the same plasmid of the mutant lacking the htrI gene as well as sopI results in production of pigment with a normal absorption spectrum but altered photochemical properties, and no phototaxis by the transformants. Analysis of flash-induced absorbance changes shows that the transducer protein increases light-induced production of the photocycle intermediate S373, the SR-I signaling conformation, and modulates the rate of S373 return to the prestimulus state, rendering this return pH-independent. These effects are interpreted in terms of receptor/transducer interactions that influence proton transfer reactions occurring in the photoactive site.

Synthesis of a gene for sensory rhodopsin I and its functional expression in Halobacterium halobium.

We have designed, synthesized, and expressed in Halobacterium halobium a gene encoding sensory rhodopsin I (SR-I). The gene has been optimized for cassette mutagenesis by incorporating 30 unique restriction sites with uniform spacing throughout the 720-bp coding region. For expression, the coding region was placed downstream of the promoter and translation initiation region of the bacterioopsin gene on a selectable vector. This construct encodes SR-I with an extended N terminus that includes the 13-amino acid leader sequence and the 8-amino acid N terminus of bacterioopsin. To obtain a SR-I- H. halobium strain for expressing the synthetic gene, we used homologous recombination to delete the chromosomal gene encoding SR-I, sopI. The deletion strain was transformed with the synthetic sopI expression vector. Using antibody directed against the C-terminal region of SR-I, we detected in transformant membranes a protein with the electrophoretic mobility expected for SR-I with a processed N-terminal extension. The synthetic gene product was functionally identical to SR-I. Its flash-induced absorption difference spectrum and photochemical reaction cycle in membrane envelope vesicles were characteristic of SR-I. The protein fully restored phototaxis responses in the deletion strain.

Conformational changes in sensory rhodopsin I: similarities and differences with bacteriorhodopsin, halorhodopsin, and rhodopsin.

FTIR difference spectra have been obtained for the sR587----S373 phototransition of sensory rhodopsin I (sR-I), a signal-transducing protein of Halobacterium halobium. The vibrational modes of the sR587 chromophore have frequencies close to those of the bacteriorhodopsin bR568 chromophore, confirming that the two chromophores have very similar structures and environments. However, the sR-I Schiff base C = N stretch frequency is downshifted relative to bR, consistent with weaker hydrogen bonding with its counterion(s). The carboxyl (COOH) stretch modes of sR-I and halorhodopsin (hR) are at the same frequencies. On the basis of sequence homologies, these bands can be assigned to Asp-106 in helix D and/or Asp-201 in helix G. In contrast, no band was found that could be assigned to the protonation of Asp-76. In bR, the homologous residue Asp-85 serves as the acceptor group for the Schiff base proton. Bands appear in the amide I and II regions at similar frequencies in sR-I, hR, and bR, indicating that despite their different functions they all undergo closely related structural changes. Bands are also detected in the C-H stretch region, possibly due to alterations in the membrane lipids. Similar spectral features are also observed in the lipids of rhodopsin-containing photoreceptor membrane upon light activation.