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PubMed1984

Halide binding by the purified halorhodopsin chromoprotein. I. Effects on the chromophore.

The halorhodopsin chromoprotein, a retinal-protein complex with an apparent molecular mass of 20 kilo-daltons, exhibits all of the halide-dependent effects found for the chromophore of functional halorhodopsin in cell envelope vesicles. With increasing halide concentration (a) an alkali-dependent 580/410 nm chromophore equilibrium (attributed to reversible deprotonation of the retinal Schiff's base) is shifted toward the 580-nm chromophore and (b) the flash-induced photocycle proceeds increasingly via P520, rather than via P660. The halide-binding site(s) responsible for these effects must reside, therefore, in the chromoprotein. Chloride and bromide are about equivalent, but iodide is much less effective in these effects and in being transported. Several other anions, i.e. thiocyanate, nitrate, phosphate, and acetate, affect the absorption maximum of the chromophore but do not allow the production of P520 upon flash illumination and are not transported. However, these ions appear to compete with chloride in the flash experiments. These observations suggest that binding of anions to a relatively nonspecific site affects the protonation state of the Schiff's base in the chromophore. Either this site directly or a more specific site, connected to the first one by a sequential pathway, is involved with the photocycle intermediates and with chloride transport by halorhodopsin.

Anions↗
PubMed1984

Halide binding by the purified halorhodopsin chromoprotein. II. New chloride-binding sites revealed by 35Cl NMR.

Halorhodopsin is a light-driven chloride pump in the cell membrane of Halobacterium halobium. Recently, a polypeptide of apparent Mr = 20,000 has been purified that contains the halorhodopsin chromophore. Here we use 35Cl NMR to show that the purified chromoprotein possesses two previously unknown classes of chloride-binding sites. One class exhibits a low affinity (KD much greater than 1 M) for chloride and bromide. The second class exhibits a higher affinity (KD = 110 +/- 50 mM) for chloride and also binds other anions according to the affinity series I-, SCN- greater than Br-, NO-3 greater than Cl- greater than F-, citrate. Both classes of NMR site remain intact at pH 11, indicating that the essential positive charges are provided by arginine. Also, both classes are unaffected by bleaching, suggesting that the sites are not in the immediate vicinity of the halorhodopsin chromophore. Although the chromoprotein also appears to contain the chloride-transport site (Steiner, M., Oesterhelt, D., Ariki, M., and Lanyi, J. K. (1984) J. Biol. Chem. 259, 2179-2184), this site was not detected by 35Cl NMR, suggesting that the transport site is in the interior of the protein where it is sampled slowly by chloride in the medium. It is proposed that the purified chromoprotein possesses a channel leading from the medium to the transport site and that the channel contains the high affinity NMR site which facilitates the migration of chloride between the medium and the transport site. We have also used 35Cl NMR to study chloride binding to purified monomeric bacteriorhodopsin; however, this protein contains no detectable chloride-binding sites.

Bacteriorhodopsins↗
PubMed1982

Identification of the retinal-binding protein in halorhodopsin.

Cell envelope vesicles prepared from a retinal-deficient strain of Halobacterium halobium contained the apoprotein of halorhodopsin, but not the apoprotein of bacteriorhodopsin. Halorhodopsin was reconstituted in these membranes with tritium-labeled retinal and the preparation was reduced with sodium cyanoborohydride. The product was a bleached pigment in which the retinal-protein bond was resistant to hydroxylamine cleavage. Fluorography of sodium dodecyl sulfate/urea-polyacrylamide gels showed that one protein was radioactively labeled, almost exclusively. This protein migrated with an apparent molecular weight somewhat lower than that of bacteriorhodopsin, which was reconstituted in membranes from another strain and radioactively labeled under the same conditions. Thus, the retinal-binding component of halorhodopsin is a small protein, with an apparent molecular weight of approximately 25,000.

Bacteriorhodopsins↗
PubMed1993

Projection structure of halorhodopsin from Halobacterium halobium at 6 A resolution obtained by electron cryo-microscopy.

Two-dimensional crystals of halorhodopsin (HR), in space group p42(1)2 (a = 102 A) have been obtained using the overexpressing Halobacterium halobium strain D2. An HR membrane fraction with the same buoyant density as purple membrane (HR-PM) was obtained by homogenization and sucrose gradient purification and used for electron cryomicroscopic analysis. Electron micrographs and electron diffraction patterns of HR-PM were recorded at liquid nitrogen temperatures. The micrographs showed significant diffraction out to 9 A resolution optically and to 6 A after computer processing. By combining data from electron micrographs and electron diffraction patterns, a projection map of HR was calculated. The crystal form of the isolated HR consists of one membrane in which alternating halorhodopsin tetramers are oriented in opposite directions across the membrane. It is not known whether this occurs by misinsertion of some of the molecules in vivo, or by adventitious fusion at some point during isolation. The projected structure of the HR molecule to a resolution of 6A is almost identical to that found for bacteriorhodopsin (BR). This physical structural similarity thus complements the known sequence relatedness to BR.

Bacteriorhodopsins↗
PubMed1995

Three-dimensional structure of halorhodopsin at 7 A resolution.

Two-dimensional crystalline patches containing the light-driven chloride pump, halorhodopsin, appear to form spontaneously in the cell membrane of an overproducing strain of Halobacterium. The three-dimensional structure (space group p42(1)2, a = 102 A) has been analysed by electron cryo-microscopy of tilted specimens. The map shows that halorhodopsin (HR) has an arrangement of seven transmembrane helices similar to that found in the related proton pump bacteriohodopsin (BR). The orientation of the polypeptide framework of HR in the membrane is rotated by 3 degrees relative to BR about an axis in the plane and the intramolecular space between the helices BC FG, which line the cytoplasmic half channel, appears slightly larger in HR than in BR, as would be expected for a chloride channel. The crystals of HR were too small for electron diffraction analysis of tilted specimens, so both the amplitudes and the phases of the Fourier components were obtained from images. This required anisotropic scaling of the image amplitudes in addition to correction for the defocus phase contrast transfer function. The procedure of rescaling the data (in this case roughly equivalent to sharpening with a temperature factor of-490) to compensate for a variety of image and crystal defects may also prove useful in the analysis of other structures for which no prior knowledge of a homologous structure exists and for which only small crystals can be obtained.

Amino Acid Sequence↗
PubMed2001

Direct observation of different surface structures on high-resolution images of native halorhodopsin.

Halorhodopsin (HR) was investigated with atomic force microscopic techniques (AFM) in aqueous solution. Two-dimensional (2D) crystals of HR were obtained by purifying an HR membrane fraction with the same buoyant density as the purple membrane (HR-PM) from the overexpressing strain Halobacterium salinarum D2. The membrane patches of HR were immobilized on mica. Images with a resolution up to 14 A were recorded. Crystals showed an orthogonal structure and the orientation of the molecules showed p42(1)2 symmetry; thus, alternate tetramers are inverted in the membrane. The crystal surface was found to display different structures depending on the imaging force used, indicating that some parts of the HR molecule are more rigid but others more compressible. From samples with single tetramers missing in the crystalline patches dimensions of the unit cell could be determined. Helix-connecting loops in single molecules of halorhodopsin were assigned. The images indicate that the large extracellular BC loop covers the whole molecule and is very flexible.

Aluminum Silicates↗
PubMed1986

Effects of arginine modification on the photocycle of halorhodopsin.

Exhaustive reaction with phenylglyoxal removed 9 of the 12 arginine and 1 of the 2 lysine residues in detergent-solubilized halorhodopsin, without affecting the chromophore. The consequences of this extensive removal of positive charges on various chloride-binding equilibria and the photochemistry were evaluated. No significant effects were seen on the affinity of Site I to chloride and on the increase in the pKa of Schiff-base deprotonation, which is caused by the chloride binding at this site. No significant effects were seen on the affinity of Site II to chloride, either. However, the photocycle of the pigment was affected. Kinetic modeling of the observed changes in flash-induced absorption changes suggests that the modification increases the affinity of the main halorhodopsin photointermediate to chloride by about fourfold. If chloride translocation involves release of chloride from this intermediate during the transport cycle, the result might explain the observed partial inhibitory effects on chloride transport. Plausible models of chloride translocation include reversible binding of the anion by positively charged groups, strategically arranged in the protein. The results indicate that two of the three spectroscopically observable chloride-dependent equilibria do not depend on a large number of positively charged residues in the protein. To the extent that the unaffected equilibria represent association and dissociation which occur during chloride translocation, at least part of the chloride translocation might be accomplished with the participation of only a few positively charged residues.

Arginine↗
PubMed1984

Nature of the principal photointermediate of halorhodopsin.

Two alternative hypotheses have been presented as to the nature of the principal halorhodopsin photointermediate: a) it is a form whose its absorption band is shifted from the 575 nm position to 500 or 520 nm, and b) it is a form whose absorption band is shifted to only about 565 nm, but with an altered band shape so it exhibits a fortuitous difference peak near 500 nm. Such a shift with a maximum near 500 nm is also obtained in the dark when chloride is removed from the sample, suggesting the hypothesis that the spectral changes reflect the transient detachment of chloride from a binding site (Ogurusu et al, J. Biochem. Tokyo 95, 1073-1082, 1984). Comparison of the quantum yields of flash-induced absorption changes in halorhodopsin and bacteriorhodopsin strongly suggests, however, that hypothesis b) is untenable.

Bacteriorhodopsins↗
PubMed1990

Anion binding to the chloride pump, halorhodopsin, and its implications for the transport mechanism.

The light-driven chloride pump, halorhodopsin, binds and transports chloride across the membrane, and to a lesser extent nitrate. Binding and transport kinetics, and resonance Raman spectra of the retinal Schiff base, with these anions suggest the existence of two mutually exclusive binding sites. One of these may be the uptake site, and the other the release site during the transport. Plausible locations can be suggested for these sites, because halorhodopsin is a small protein with few buried positively charged residues, and the primary structure of a second pigment with similar function has recently become available for comparison.

Bacteriorhodopsins↗
PubMed2001

Halide dependence of the halorhodopsin photocycle as measured by time-resolved infrared spectra.

Time-resolved Fourier transform infrared (FTIR) difference spectra of the halorhodopsin (hR) photocycle have been collected from 3 micros to 100 ms in saturating concentrations of KCl or KBr. Kinetic analysis of these data revealed two decay processes, with time constants of tau(1) approximately 150 micros and tau(2) approximately 16 ms in the presence of either halide, with tau(2) describing the return to the starting (hR) state. Comparison to previous low-temperature FTIR spectra of hR intermediates confirms that characteristic hK and hL spectral features are both present before the tau(1) decay, in a state previously defined as hK(L) (Dioumaev, A., and M. Braiman. 1997. Photochem. Photobiol. 66:755-763). However, the relative sizes of these features depend on which halide is present. In Br-, the hL features are clearly more dominant than in Cl-. Therefore, the state present before tau(1) is probably best described as an hK(L)/hL(1) equilibrium, instead of a single hK(L) state. Different halides affect the relative amounts of hK(L) and hL(1) present, i.e., Cl- produces a much more significant back-reaction from hL(1) to hK(L) than does Br-. The halide dependence of this back-reaction could therefore explain the halide selectivity of the halorhodopsin anion pump.

Bacteriorhodopsins↗
PubMed2003

Ser-130 of Natronobacterium pharaonis halorhodopsin is important for the chloride binding.

Pharaonis halorhodopsin (phR) is an inward light-driven chloride ion pump from Natronobacterium pharaonis. In order to clarify the role of Ser-130(phR) residue which corresponds to Ser-115(shR) for salinarum hR on the anion-binding affinity, the wild-type and Ser-130 mutants substituted with Thr, Cys and Ala were expressed in E. coli cells and solubilized with 0.1% n-dodecyl beta-D-maltopyranoside The absorption maximum (lambda(max)) of the S130T mutant indicated a blue shift from that of the wild type in the absence and presence of chloride. For S130A, a large red shift (12 nm) in the absence of chloride was observed. The wild-type and all mutants showed the blue-shift of lambda(max) upon Cl(-) addition, from which the dissociation constants of Cl(-) were determined. The dissociation constants were 5, 89, 153 and 159 mM for the wild-type, S130A, S130T and S130C, respectively, at pH 7.0 and 25 degrees C. Circular dichroic spectra of the wild-type and the Ser-130 mutants exhibited an oligomerization. The present study revealed that the Ser-130 of N. pharaonis halorhodopsin is important for the chloride binding.

Amino Acid Sequence↗
PubMed2002

Halorhodopsin: light-driven ion pumping made simple?

Halorhodopsin, a light-driven halide pump, is the second archaeal rhodopsin involved in ion pumping to be studied at high resolution by X-ray crystallography. Like its cousin bacteriorhodopsin, halorhodopsin couples vectorial ion transport to the isomerisation state of a covalently linked retinal. Given the similarity and interconvertability of these two ion pumps, a unified mechanism for ion translocation by archaeal rhodopsins is now emerging.

Biological Transport, Active↗
PubMed1994

Blue halorhodopsin from Natronobacterium pharaonis: wavelength regulation by anions.

Halorhodopsin, the chloride pump from Natronobacterium pharaonis (pharaonis hR), was isolated under conditions of low ionic strength. The quotient between the optical densities of pharaonis hR in 4 M NaCl at 280 and 577 nm amounts to 1.1, indicating a high purity of the protein and integrity of the chromophore. Gel filtration chromatography of the purified pharaonos hR at neutral pH and in the absence of inorganic salts leads to a shift to the absorption maximum to 600 nm (pharaonis hRblue). The purple color can be regained by the addition of anions such as chloride, iodide, azide, nitrate, and also fluoride. The absorption maxima are dependent on the nature of the anion and the pH. At pH 7, sulfate does not influence the 600-nm absorption, while at pH 4.5 the absorption maximum is shifted to 581 nm. The blue form of halorhodopsin (pharaonis hRblue) was titrated with different sodium salts, such as chloride, azide, and nitrate. The half-maximal binding is in the millimolar range, with Br < Cl < NO3 < N3 < BrO3. Deprotonation of the Schiff base can be accomplished by treatment of pharaonis hR or pharaonis hRblue with base. The apparent pK of the Schiff base in pharaonis hRblue was determined to be 8.5. The pK shifted to 8.0 in the presence of 150 mM Na2SO4, whereas the pK of pharaonis hR in 150 mM NaCl is 9.6. In the photocycle of the chloride- and nitrate-containing pharaonis hR, a species similar to hR520 could be detected. Pharaonis hR reconstituted with azide shows photochemical reactions similar to the photocycle of bacteriorhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)

Anions↗
PubMed1986

Secondary structure of halorhodopsin.

Ultraviolet circular dichroism (CD) spectroscopy in the interval from 190 to 240 nm has been used to estimate the secondary structural content of halorhodopsin (hR), a light-driven chloride pump isolated from the membranes of Halobacterium halobium. Least-squares curve fitting of the CD spectrum for hR solubilized with octyl glucoside yields an alpha-helical content of approximately 50% and a beta-structure content of approximately 30%. The CD spectrum of hR is unaffected by the absence or presence of chloride ions or by the ionic strength of the medium. The CD spectrum of halorhodopsin is very similar to that of bacteriorhodopsin, indicating that these light-driven pumps possess nearly identical fractions of alpha- and beta-secondary structures.

Bacteriorhodopsins↗
PubMed1987

Structure of the retinal chromophore in the hRL intermediate of halorhodopsin from resonance raman spectroscopy.

Time-resolved resonance Raman spectra of the hRL intermediate of halorhodopsin have been obtained. The structurally sensitive fingerprint region of the hRL spectrum is very similar to that of bacteriorhodopsin's L550 intermediate, which is known to have a 13-cis configuration. This indicates that hRL contains a 13-cis chromophore and that an all-trans----13-cis isomerization occurs in the halorhodopsin photocycle. hRL exhibits a Schiff base stretching mode at 1644 cm-1, which shifts to 1620 cm-1 in D2O. This demonstrates that the Schiff base linkage to the protein is protonated. The insensitivity of the C-C stretching mode frequencies to N-deuteriation suggests that the Schiff base configuration is anti. The 24 cm-1 shift of the Schiff base mode in D2O indicates that the Schiff base proton in hRL has a stronger hydrogen-bonding interaction with the protein than does hR578.

Bacteriorhodopsins↗
PubMed2005

Effect of anions on the photocycle of halorhodopsin. Substitution of chloride with formate anion.

Halorhodopsin from Natronomonas pharaonis is a light-driven chloride pump which transports a chloride anion across the plasma membrane following light absorption by a retinal chromophore which initiates a photocycle. It was shown that the chloride anion bound in the vicinity of retinal PSB can be replaced by several inorganic anions, including azide which converts the chloride pump into a proton pump and induces formation of an M-like intermediate detected in the bR photocycle but not in native halorhodopsin. Here we have studied the possibility of replacing the chloride anion with organic anions and have followed the photocycle under several conditions. It is revealed that the chloride can be replaced with a formate anion but not with larger organic anions such as acetate. Flash photolysis experiments detected in the formate pigment an M-like intermediate characterized by a lifetime much longer than that of the O intermediate. The lifetime of the M-like intermediate depends on the pH, and its decay is significantly accelerated at low pH. The decay rate exhibited a titration-like curve, suggesting that the protonation of a protein residue controls the rate of M decay. Similar behavior was detected in N. pharaonis pigments in which the chloride anion was replaced with NO(2)(-) and OCN(-) anions. It is suggested that the formation of the M-like intermediate indicates branching pathways from the L intermediate or basic heterogeneity in the original pigment.

Acetates↗
PubMed1997

Chromophore-anion interactions in halorhodopsin from Natronobacterium pharaonis probed by time-resolved resonance Raman spectroscopy.

Halorhodopsin of Natronobacterium pharaonis which acts as a light-driven chloride pump is studied by time-resolved resonance Raman spectroscopy. In single-beam experiments, resonance Raman spectra were obtained of the parent state HR578 and the first thermal intermediate HR520. The parent state is structural heterogeneous including ca. 80% all-trans and 20% 13-cis isomers. The resonance Raman spectra indicate that the all-trans conformer exhibits essentially the same chromophoric structure as in the parent states of bacteriorhodopsin or halorhodopsin from Halobacterium salinarium. Special emphasis of the resonance Raman spectroscopic analysis was laid on the C=C and C=N stretching region in order to probe the interactions between the protonated Schiff base and various bound anions (chloride, bromide, iodide). These investigations were paralleled by spectroscopic studies of retinal Schiff base model complexes in different solvents in an attempt to determine the various parameters which control the C=C and C=N stretching frequencies. From these data, it was concluded that in the parent state the anion is not involved in hydrogen bonding interactions with the Schiff base proton but is presumably bound to a nearby (positively charged) amino acid residue. On the other hand, the anion still exerts an appreciable effect on the chromophore structure which is, for instance, reflected by the variation of the isomer composition in the presence of different anions and in the anion-depleted form. In contrast to the parent state, the intermediate HR520 reveals frequency shifts of the C=N stretching in the presence of different anions. These findings indicate a closer proximity of the bound anion to the Schiff base proton which is sufficient for hydrogen bonding interactions. These changes of the anion-chromophore interaction upon transition from HR578 to HR520 may be related to the coupling of the chromophore movement with the anion translocation.

Anions↗
PubMed1999

Existence of two L photointermediates of halorhodopsin from Halobacterium salinarium, differing in their protein and water FTIR bands.

FTIR difference spectra were recorded for the photoreactions of halorhodopsin from Halobacterium salinarium at 170 and 250 K. Obvious differences at the two temperatures were noted in neither the visible spectra nor the FTIR bands of the chromophore. However, perturbation of Asp141 is observed in the L intermediate at 250 K but not at 170 K. We named these photoproducts La (at 170 K) and Lb (at 250 K). The spectrum of Lb is distinct from that of La also in the different shifts of water O-H stretching bands, and larger changes in the bands from the protein backbone with different sensitivities to varying the halide. These results suggest that the photocycle of halorhodopsin contains two L states, La and Lb, in which the structure of protein and internal water molecules is different but chloride stays at the same site close to the Schiff base.

Bacteriorhodopsins↗