Structural characteristics around the β-ionone ring of the retinal chromophore in Salinibacter sensory rhodopsin I

Organisms sense and respond to environmental stimuli through membrane-embedded receptors and transducers. Sensory rhodopsin I (SRI) and sensory rhodopsin II (SRII) are the photoreceptors for the positive and negative phototaxis in microorganisms, respectively. They form signaling complexes in the membrane with their cognate transducer proteins, HtrI and HtrII, and these SRI-HtrI and SRII-HtrII complexes transmit a light signal through their cytoplasmic sensory signaling system, inducing opposite effects (i.e., the inactivation or activation of the kinase CheA). Here we found, by using Fourier transformed infrared spectroscopy, that a conserved residue, Asp102 in Salinibacter SRI (SrSRI), which is located close to the β-ionone ring of the retinal chromophore, is deprotonated upon formation of the active M-intermediate. Furthermore, the D102E mutant of SrSRI affects the structure and/or structural changes of Cys130. This mutant shows a large spectral shift and is comparably unstable, especially in the absence of Cl(-). These phenomena have not been observed in the wild-type, or the N105Q and N105D mutants of Natronomonas pharaonis SRII (NpSRII), indicating differences in the structure and structural changes between SrSRI and NpSRII around the β-ionone ring. These differences could also be supported by the measurements of the reactivity with the water-soluble reagent azide. On the basis of these results, we discuss the structure and structural changes around the retinal chromophore in SrSRI.     

Structural changes of Salinibacter sensory rhodopsin I upon formation of the K and M photointermediates

Sensory rhodopsin I (SRI) is one of the most interesting photosensory receptors in nature because of its ability to mediate opposite signals depending on light color by photochromic one-photon and two-photon reactions. Recently, we characterized SRI from eubacterium Salinibacter ruber (SrSRI). This protein allows more detailed information about the structure and structural changes of SRI during its action to be obtained. In this paper, Fourier transform infrared (FTIR) spectroscopy is applied to SrSRI, and the spectral changes upon formation of the K and M intermediates are compared with those of other archaeal rhodopsins, SRI from Halobacterium salinarum (HsSRI), sensory rhodopsin II (SRII), bacteriorhodopsin (BR), and halorhodopsin (HR). Spectral comparison of the hydrogen out-of-plane (HOOP) vibrations of the retinal chromophore in the K intermediates shows that extended choromophore distortion takes place in SrSRI and HsSRI, as well as in SRII, whereas the distortion is localized in the Schiff base region in BR and HR. It appears that sensor and pump functions are distinguishable from the spectral feature of HOOP modes. The HOOP band at 864 cm(-1) in SRII, important for negative phototaxis, is absent in SrSRI, suggesting differences in signal transfer mechanism between SRI and SRII. The strongly hydrogen-bound water molecule, important for proton pumps, is observed at 2172 cm(-1) in SrSRI, as well as in BR and SRII. The formation of the M intermediate accompanies the appearance of peaks at 1753 (+) and 1743 (-) cm(-1), which can be interpreted as the protonation signal of the counterion (Asp72) and the proton release signal from an unidentified carboxylic acid, respectively. The structure and structural changes of SrSRI are discussed on the basis of the present infrared spectral comparisons with other rhodopsins.     

Salinibacter sensory rhodopsin: sensory rhodopsin I-like protein from a eubacterium

Halobacterium salinarum sensory rhodopsin I (HsSRI), a dual receptor regulating both negative and positive phototaxis in haloarchaea, transmits light signals through changes in protein-protein interactions with its transducer, halobacterial transducer protein I (HtrI). Haloarchaea also have another sensor pigment, sensory rhodopsin II (SRII), which functions as a receptor regulating negative phototaxis. Compared with HsSRI, the signal relay mechanism of SRII is well characterized because SRII from Natronomonus pharaonis (NpSRII) is much more stable than HsSRI and HsSRII, especially in dilute salt solutions and is much more resistant to detergents. Two genes encoding SRI homologs were identified from the genome sequence of the eubacterium Salinibacter ruber. Those sequences are distantly related to HsSRI ( approximately 40% identity) and contain most of the amino acid residues identified as necessary for its function. To determine whether those genes encode functional protein(s), we cloned and expressed them in Escherichia coli. One of them (SrSRI) was expressed well as a recombinant protein having all-trans retinal as a chromophore. UV-Vis, low-temperature UV-Vis, pH-titration, and flash photolysis experiments revealed that the photochemical properties of SrSRI are similar to those of HsSRI. In addition to the expression system, the high stability of SrSRI makes it possible to prepare large amounts of protein and enables studies of mutant proteins that will allow new approaches to investigate the photosignaling process of SRI-HtrI.     

Direct observation of the structural change of Tyr174 in the primary reaction of sensory rhodopsin II

Sensory rhodopsin II (SRII) is a negative phototaxis receptor containing retinal as its chromophore, which mediates the avoidance of blue light. The signal transduction is initiated by the photoisomerization of the retinal chromophore, resulting in conformational changes of the protein which are transmitted to a transducer protein. To gain insight into the SRII sensing mechanism, we employed time-resolved ultraviolet resonance Raman spectroscopy monitoring changes in the protein structure in the picosecond time range following photoisomerization. We used a 450 nm pump pulse to initiate the SRII photocycle and two kinds of probe pulses with wavelengths of 225 and 238 nm to detect spectral changes in the tryptophan and tyrosine bands, respectively. The observed spectral changes of the Raman bands are most likely due to tryptophan and tyrosine residues located in the vicinity of the retinal chromophore, i.e., Trp76, Trp171, Tyr51, or Tyr174. The 225 nm UVRR spectra exhibited bleaching of the intensity for all the tryptophan bands within the instrumental response time, followed by a partial recovery with a time constant of 30 ps and no further changes up to 1 ns. In the 238 nm UVRR spectra, a fast recovering component was observed in addition to the 30 ps time constant component. A comparison between the spectra of the WT and Y174F mutant of SRII indicates that Tyr174 changes its structure and/or environment upon chromophore photoisomerization. These data represent the first real-time observation of the structural change of Tyr174, of which functional importance was pointed out previously.     

Environment around the chromophore in pharaonis phoborhodopsin: mutation analysis of the retinal binding site.

Phoborhodopsin (pR or sensory rhodopsin II, sRII) and pharaonis phoborhodopsin (ppR or pharaonis sRII, psRII) have a unique absorption maximum (lambda(max)) compared with three other archaeal rhodopsins: lambda(max) of pR and ppR is approx. 500 nm and of others (e.g. bacteriorhodopsin, bR) is 560-590 nm. To determine the residue contributing to the opsin shift from ppR to bR, we constructed various ppR mutants, in which a single residue was substituted for a residue corresponding to that of bR. The residues mutated were those which differ from that of bR and locate within 5 A from the conjugated polyene chain of the chromophore or any methyl group of the polyene chain. The shifts of lambda(max) of all mutants were small, however. We constructed a mutant in which all residues which differ from those of bR in the retinal binding site were simultaneously substituted for those of bR, but the shift was only from 499 to 509 nm. Next, we constructed a mutant in which 10 residues located within 5 A from the polyene as described above were simultaneously substituted. Only 44% of the opsin shift (lambda(max) of 524 nm) from ppR to bR was obtained even when all amino acids around the chromophore were replaced by the same residues as bR. We therefore conclude that the structural factor is more important in accounting for the difference of lambda(max) between ppR and bR rather than amino acid substitutions. The possible structural factors are discussed.     

A microbial rhodopsin with a unique retinal composition shows both sensory rhodopsin II and bacteriorhodopsin-like properties

Rhodopsins possess retinal chromophore surrounded by seven transmembrane α-helices, are widespread in prokaryotes and in eukaryotes, and can be utilized as optogenetic tools. Although rhodopsins work as distinctly different photoreceptors in various organisms, they can be roughly divided according to their two basic functions, light-energy conversion and light-signal transduction. In microbes, light-driven proton transporters functioning as light-energy converters have been modified by evolution to produce sensory receptors that relay signals to transducer proteins to control motility. In this study, we cloned and characterized two newly identified microbial rhodopsins from Haloquadratum walsbyi. One of them has photochemical properties and a proton pumping activity similar to the well known proton pump bacteriorhodopsin (BR). The other, named middle rhodopsin (MR), is evolutionarily transitional between BR and the phototactic sensory rhodopsin II (SRII), having an SRII-like absorption maximum, a BR-like photocycle, and a unique retinal composition. The wild-type MR does not have a light-induced proton pumping activity. On the other hand, a mutant MR with two key hydrogen-bonding residues located at the interaction surface with the transducer protein HtrII shows robust phototaxis responses similar to SRII, indicating that MR is potentially capable of the signaling. These results demonstrate that color tuning and insertion of the critical threonine residue occurred early in the evolution of sensory rhodopsins. MR may be a missing link in the evolution from type 1 rhodopsins (microorganisms) to type 2 rhodopsins (animals), because it is the first microbial rhodopsin known to have 11-cis-retinal similar to type 2 rhodopsins.     

Electrostatic potential at the retinal of three archaeal rhodopsins: implications for their different absorption spectra

The color tuning mechanism of the rhodopsin protein family has been in the focus of research for decades. However, the structural basis of the tuning mechanism in general and of the absorption shift between rhodopsins in particular remains under discussion. It is clear that a major determinant for spectral shifts between different rhodopsins are electrostatic interactions between the chromophore retinal and the protein. Based on the Poisson-Boltzmann equation, we computed and compared the electrostatic potential at the retinal of three archaeal rhodopsins: bacteriorhodopsin (BR), halorhodopsin (HR), and sensory rhodopsin II (SRII) for which high-resolution structures are available. These proteins are an excellent test case for understanding the spectral tuning of retinal. The absorption maxima of BR and HR are very similar, whereas the spectrum of SRII is considerably blue shifted--despite the structural similarity between these three proteins. In agreement with their absorption maxima, we find that the electrostatic potential is similar in BR and HR, whereas significant differences are seen for SRII. The decomposition of the electrostatic potential into contributions of individual residues, allowed us to identify seven residues that are responsible for the differences in electrostatic potential between the proteins. Three of these residues are located in the retinal binding pocket and have in fact been shown to account for part of the absorption shift between BR and SRII by mutational studies. One residue is located close to the beta-ionone ring of retinal and the remaining three residues are more than 8 A away from the retinal. These residues have not been discussed before, because they are, partly because of their location, no obvious candidates for the spectral shift among BR, HR, and SRII. However, their contribution to the differences in electrostatic potential is evident. The counterion of the Schiff base, which is frequently discussed to be involved in the spectral tuning, does not contribute to the dissimilarities between the electrostatic potentials.     

Engineering a functional blue-wavelength-shifted rhodopsin mutant

We report an effort to engineer a functional, maximally blue-wavelength-shifted version of rhodopsin. Toward this goal, we first constructed and assayed a number of previously described mutations in the retinal binding pocket of rhodopsin, G90S, E122D, A292S, and A295S. Of these mutants, we found that only mutants E122D and A292S were like the wild type (WT). In contrast, mutant G90S exhibited a perturbed photobleaching spectrum, and mutant A295S exhibited decreased ability to activate transducin. We also identified and characterized a new blue-wavelength-shifting mutation (at site T118), a residue conserved in most opsin proteins. Interestingly, although residue T118 contacts the critically important C9-methyl group of the retinal chromophore, the T118A mutant exhibited no significant perturbation other than the blue-wavelength shift. In analyzing these mutants, we found that although several mutants exhibited different rates of retinal release, the activation energies of the retinal release were all approximately 20 kcal/mol, almost identical to the value found for WT rhodopsin. These latter results support the theory that chemical hydrolysis of the Schiff base is the rate-limiting step of the retinal release pathway. A combination of the functional blue-wavelength-shifting mutations was then used to generate a triple mutant (T118A/E122D/A292S) which exhibited a large blue-wavelength shift (absorption lambda(max) = 453 nm) while exhibiting minimal functional perturbation. Mutant T118A/E122D/A292S thus offers the possibility of a rhodopsin protein that can be worked with and studied using more ambient lighting conditions, and facilitates further study by fluorescence spectroscopy.     

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.     

Function of extracellular loop 2 in rhodopsin: glutamic acid 181 modulates stability and absorption wavelength of metarhodopsin II

The second extracellular loop of rhodopsin folds back into the membrane-embedded domain of the receptor to form part of the binding pocket for the 11-cis-retinylidene chromophore. A carboxylic acid side chain from this loop, Glu181, points toward the center of the retinal polyene chain. We studied the role of Glu181 in bovine rhodopsin by characterizing a set of site-directed mutants. Sixteen of the 19 single-site mutants expressed and bound 11-cis-retinal to form pigments. The lambda(max) value of mutant pigment E181Q showed a significant spectral red shift to 508 nm only in the absence of NaCl. Other substitutions did not significantly affect the spectral features of the mutant pigments in the dark. Thus, Glu181 does not contribute significantly to spectral tuning of the ground state of rhodopsin. The most likely interpretation of these data is that Glu181 is protonated and uncharged in the dark state of rhodopsin. The Glu181 mutants displayed significantly increased reactivity toward hydroxylamine in the dark. The mutants formed metarhodopsin II-like photoproducts upon illumination but many of the photoproducts displayed shifted lambda(max) values. In addition, the metarhodopsin II-like photoproducts of the mutant pigments had significant alterations in their decay rates. The increased reactivity of the mutants to hydroxylamine supports the notion that the second extracellular loop prevents solvent access to the chromophore-binding pocket. In addition, Glu181 strongly affects the environment of the retinylidene Schiff base in the active metarhodopsin II photoproduct.     

Steric constraint in the primary photoproduct of sensory rhodopsin II is a prerequisite for light-signal transfer to HtrII

Sensory rhodopsin II (SRII, also called pharaonis phoborhodopsin, ppR) is responsible for negative phototaxis in Natronomonas pharaonis. Photoisomerization of the retinal chromophore from all- trans to 13- cis initiates conformational changes in the protein, leading to activation of the cognate transducer protein (HtrII). We previously observed enhancement of the C 14-D stretching vibration of the retinal chromophore at 2244 cm (-1) upon formation of the K state and interpreted that a steric constraint occurs at the C 14D group in SRII K. Here, we identify the counterpart of the C 14D group as Thr204, because the C 14-D stretching signal disappeared in T204A, T204S, and T204C mutants as well as a C 14-HOOP (hydrogen out-of-plane) vibration at 864 cm (-1). Although the K state of the wild-type bacteriorhodopsin (BR), a light-driven proton pump, possesses neither 2244 nor 864 cm (-1) bands, both signals appeared for the K state of a triple mutant of BR that functions as a light sensor (P200T/V210Y/A215T). We found a positive correlation between these vibrational amplitudes of the C 14 atom at 77 K and the physiological phototaxis response. These observations strongly suggest that the steric constraint between the C 14 group of retinal and Thr204 of the protein is a prerequisite for light-signal transduction by SRII.     

Early photocycle structural changes in a bacteriorhodopsin mutant engineered to transmit photosensory signals

Bacteriorhodopsin (BR) and sensory rhodopsin II (SRII) function as a light-driven proton pump and a receptor for negative phototaxis in haloarchaeal membranes, respectively. SRII transmits light signals through changes in protein-protein interaction with its transducer HtrII. Recently, we converted BR by three mutations into a form capable of transmitting photosignals to HtrII to mediate phototaxis responses. The BR triple mutant (BR-T) provides an opportunity to identify structural changes necessary to activate HtrII by comparing light-induced infrared spectral changes of BR, BR-T, and SRII. The hydrogen out-of-plane (HOOP) vibrations of the BR-T were very similar to those of SRII, indicating that they are distributed more extensively along the retinal chromophore than in BR, as in SRII. On the other hand, the bands of the protein moiety in BR-T are similar to those of BR, indicating that they are not specific to photosensing. The alteration of the O-H stretching vibration of Thr-204 in SRII, which we had previously shown to be essential for signal relay to HtrII, occurs also in BR-T. In addition, 1670(+)/1664(-) cm(-1) bands attributable to a distorted alpha-helix were observed in BR-T in a HtrII-dependent manner, as is seen in SRII. Thus, we identified similarities and dissimilarities of BR-T to BR and SRII. The results suggest signaling function of the structural changes of the HOOP vibrations, the O-H stretching vibration of the Thr-215 residue, and a distorted alpha-helix for the signal generation. We also succeeded in measurements of L minus initial state spectra of BR-T, which are the first FTIR spectra of L intermediates among sensory rhodopsins.     

Serine 85 in transmembrane helix 2 of short-wavelength visual pigments interacts with the retinylidene Schiff base counterion.

Short-wavelength cone visual pigments (SWS1) are responsible for detecting light from 350 to 430 nm. Models of this class of pigment suggest that TM2 has extensive contacts with the retinal binding pocket and stabilizes interhelical interactions. The role of TM2 in the structure-function of the Xenopus SWS1 (VCOP, lambda(max) = 427 nm) pigment was studied by replacement of the helix with that of bovine rhodopsin and also by mutagenesis of highly conserved residues. The TM2 chimera and G78D, F79L, M81E, P88T, V89S, and F90V mutants did not produce any significant spectral shift of the dark state or their primary photointermediate formed upon illumination at cryogenic temperatures. The mutant G77R (responsible for human tritanopia) was completely defective in folding, while C82A and F87T bound retinal at reduced levels. The position S85 was crucial for obtaining the appropriate spectroscopic properties of VCOP. S85A and S85T did not bind retinal. S85D bound retinal and had a wild-type dark state at room temperature and a red-shifted dark state at 45 K and formed an altered primary photointermediate. S85C absorbed maximally at 390 nm at neutral pH and at 365 nm at pH >7.5. The S85C dark state was red shifted by 20 nm at 45 K and formed an altered primary photointermediate. These data suggest that S85 is involved in a hydrogen bond with the protonated retinylidene Schiff base counterion in both the dark state and the primary photointermediate.     

FT-IR difference spectroscopy elucidates crucial interactions of sensory rhodopsin I with the cognate transducer HtrI

The phototaxis receptor sensory rhodopsin I (SRI) from Halobacterium salinarum interacts with its cognate transducer (HtrI) forming a transmembrane complex. After light excitation of the chromophore all-trans retinal, SRI undergoes structural changes that are ultimately transmitted to HtrI. The interaction of SRI with HtrI results in the closure of the receptor's proton pathway, which renders the photocycle recovery kinetics of SRI pH-independent. We demonstrate on heterologously expressed and reconstituted SRI-HtrI fusion proteins that the transmembrane part of HtrI (residues 1-52) as well as the downstream cytoplasmic part (residues 53-147) exhibit conformational changes after light excitation. The sum of these conformational changes is similar to those observed in the fusion constructs SRI-HtrI 1-71 and SRI-HtrI 1-147, which display pH-independent receptor kinetics. These results indicate the occurrence of spatially distinct conformational changes that are required for functional signal transmission. Kinetic and spectroscopic analysis of HtrI point mutants of Asn53 provides evidence that this residue is involved in the receptor-transducer interaction. We suggest that Asn53 plays a role similar to that of Asn74 of the HtrII from Natronobacterium pharaonis, the latter forming a hydrogen bond to the receptor within the membrane.     

Molecular mechanism of spectral tuning in sensory rhodopsin II.

Sensory rhodopsin II (SRII) is unique among the archaeal rhodopsins in having an absorption maximum near 500 nm, blue shifted roughly 70 nm from the other pigments. In addition, SRII displays vibronic structure in the lambda(max) absorption band, whereas the other pigments display fully broadened band maxima. The molecular origins responsible for both photophysical properties are examined here with reference to the 2.4 A crystal structure of sensory rhodopsin II (NpSRII) from Natronobacterium pharaonis. We use semiempirical molecular orbital theory (MOZYME) to optimize the chromophore within the chromophore binding site, and MNDO-PSDCI molecular orbital theory to calculate the spectroscopic properties. The entire first shell of the chromophore binding site is included in the MNDO-PSDCI SCF calculation, and full single and double configuration interaction is included for the chromophore pi-system. Through a comparison of corresponding calculations on the 1.55 A crystal structure of bacteriorhodopsin (bR), we identify the principal molecular mechanisms, and residues, responsible for the spectral blue shift in NpSRII. We conclude that the major source of the blue shift is associated with the significantly different positions of Arg-72 (Arg-82 in bR) in the two proteins. In NpSRII, this side chain has moved away from the chromophore Schiff base nitrogen and closer to the beta-ionylidene ring. This shift in position transfers this positively charged residue from a region of chromophore destabilization in bR to a region of chromophore stabilization in NpSRII, and is responsible for roughly half of the blue shift. Other important contributors include Asp-201, Thr-204, Tyr-174, Trp-76, and W402, the water molecule hydrogen bonded to the Schiff base proton. The W402 contribution, however, is a secondary effect that can be traced to the transposition of Arg-72. Indeed, secondary interactions among the residues contribute significantly to the properties of the binding site. We attribute the increased vibronic structure in NpSRII to the loss of Arg-72 dynamic inhomogeneity, and an increase in the intensity of the second excited (1)A(g)(-) -like state, which now appears as a separate feature within the lambda(max) band profile. The strongly allowed (1)B(u)(+)-like state and the higher-energy (1)A(g)(-) -like state are highly mixed in NpSRII, and the latter state borrows intensity from the former to achieve an observable oscillator strength.     

Determinants of visual pigment absorbance: role of charged amino acids in the putative transmembrane segments

I have investigated the effect on bovine rhodopsin's absorbance spectrum of charged amino acid changes in the putative membrane-spanning regions. A total of 14 site-directed mutants were constructed at 6 amino acid positions: 83, 86, 122, 134, 135, and 211. Two of these positions are occupied by charged amino acids that are conserved in all four human visual pigments (positions 134 and 135). In the four variable positions, single and double mutants were constructed to reproduce the intramembrane distribution of charged amino acids predicted for each human cone pigment. Following solubilization in digitonin and reconstitution with 11-cis-retinal, the photobleaching difference spectrum of each pigment was determined in the presence of hydroxylamine. The absorbance spectra of the mutant pigments are all surprisingly close to that of native bovine rhodopsin (lambda max = 498 nm), ruling out a significant role for these residues in spectral tuning.     

Proton circulation during the photocycle of sensory rhodopsin II.

Sensory rhodopsin II (SRII) in Halobacterium salinarum membranes is a phototaxis receptor that signals through its bound transducer HtrII for avoidance of blue-green light. In the present study we investigated the proton movements during the photocycle of SRII in the HtrII-free and HtrII-complexed form. We monitored sustained light-induced pH changes with a pH electrode, and laser flash-induced pH changes with the pH indicator pyranine using sealed membrane vesicles and open sheets containing the free or the complexed receptor. The results demonstrated that SRII takes up a proton in M-to-O conversion and releases it during O-decay. The uptake and release are from and to the extracellular side, and therefore SRII does not transport the proton across the membrane. The pH dependence of the SRII photocycle indicated the presence of a protonatable group (pK(a) approximately 7.5) in the extracellular proton-conducting path, which plays a role in proton uptake by the Schiff base in the M-to-O conversion. The extracellular proton circulation produced by SRII was not blocked by HtrII complexation, unlike the cytoplasmic proton conduction in SRI that was found in the same series of measurements to be blocked by its transducer, HtrI. The implications of this finding for current models of SRI and SRII signaling are discussed.     

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.     

Rapid release of retinal from a cone visual pigment following photoactivation

As part of the visual cycle, the retinal chromophore in both rod and cone visual pigments undergoes reversible Schiff base hydrolysis and dissociation following photobleaching. We characterized light-activated release of retinal from a short-wavelength-sensitive cone pigment (VCOP) in 0.1% dodecyl maltoside using fluorescence spectroscopy. The half-time (t(1/2)) of release of retinal from VCOP was 7.1 s, 250-fold faster than that of rhodopsin. VCOP exhibited pH-dependent release kinetics, with the t(1/2) decreasing from 23 to 4 s with the pH decreasing from 4.1 to 8, respectively. However, the Arrhenius activation energy (E(a)) for VCOP derived from kinetic measurements between 4 and 20 °C was 17.4 kcal/mol, similar to the value of 18.5 kcal/mol for rhodopsin. There was a small kinetic isotope (D(2)O) effect in VCOP, but this effect was smaller than that observed in rhodopsin. Mutation of the primary Schiff base counterion (VCOP(D108A)) produced a pigment with an unprotonated chromophore (λ(max) = 360 nm) and dramatically slowed (t(1/2) ~ 6.8 min) light-dependent retinal release. Using homology modeling, a VCOP mutant with two substitutions (S85D and D108A) was designed to move the counterion one α-helical turn into the transmembrane region from the native position. This double mutant had a UV-visible absorption spectrum consistent with a protonated Schiff base (λ(max) = 420 nm). Moreover, the VCOP(S85D/D108A) mutant had retinal release kinetics (t(1/2) = 7 s) and an E(a) (18 kcal/mol) similar to those of the native pigment exhibiting no pH dependence. By contrast, the single mutant VCOP(S85D) had an ~3-fold decreased retinal release rate compared to that of the native pigment. Photoactivated VCOP(D108A) had kinetics comparable to those of a rhodopsin counterion mutant, Rho(E113Q), both requiring hydroxylamine to fully release retinal. These results demonstrate that the primary counterion of cone visual pigments is necessary for efficient Schiff base hydrolysis. We discuss how the large differences in retinal release rates between rod and cone visual pigments arise, not from inherent differences in the rate of Schiff base hydrolysis but rather from differences in the properties of noncovalent binding of the retinal chromophore to the protein.     

A functional rhodopsin-green fluorescent protein fusion protein localizes correctly in transgenic Xenopus laevis retinal rods and is expressed in a time-dependent pattern

To study rhodopsin biosynthesis and transport in vivo, we engineered a fusion protein (rho-GFP) of bovine rhodopsin (rho) and green fluorescent protein (GFP). rho-GFP expressed in COS-1 cells bound 11-cis retinal, generating a pigment with spectral properties of rhodopsin (A(max) at 500 nm) and GFP (A(max) at 488 nm). rho-GFP activated transducin at 50% of the wild-type activity, whereas phosphorylation of rho-GFP by rhodopsin kinase was 10% of wild-type levels. We expressed rho-GFP in the rod photoreceptors of Xenopus laevis using the X. laevis principal opsin promoter. Like rhodopsin, rho-GFP localized to rod outer segments, indicating that rho-GFP was recognized by membrane transport mechanisms. In contrast, a rho-GFP variant lacking the C-terminal outer segment localization signal distributed to both outer and inner segment membranes. Confocal microscopy of transgenic retinas revealed that transgene expression levels varied between cells, an effect that is probably analogous to position-effect variegation. Furthermore, rho-GFP concentrations varied along the length of individual rods, indicating that expression levels varied within single cells on a daily or hourly basis. These results have implications for transgenic models of retinal degeneration and mechanisms of position-effect variegation and demonstrate the utility of rho-GFP as a probe for rhodopsin transport and temporal regulation of promoter function.