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Janos K. Lanyi - One of the best experts on this subject based on the ideXlab platform.
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[59] Spectrophotometric determination of Halorhodopsin in Halobacterium halobium Membranes
Methods in Enzymology, 2004Co-Authors: Janos K. LanyiAbstract:Publisher Summary This chapter discusses the spectrophotometric determination of Halorhodopsin in Halobacterium halobium membranes. It is reported that the high turbidity of the cell envelope preparations and their high cytochrome and low Halorhodopsin content preclude a direct spectrophotometric demonstration of the pigment, thus steps are presented in the chapter for decreasing turbidity, increase yield of Halorhodopsin. For spectrometry, the vesicle preparation in 4 M NaCl is divided into two portions and placed into 1-cm path length cuvettes, trans-Retinal, dissolved in methanol, 15 is added to the sample cuvette, and the same volume of methanol is added to the reference cuvette. Retinal reconstitution in such preparations takes 4-6 hr at room temperature, and overnight incubation with the retinal is required. Difference spectra obtained in this way show the appearance of an absorption band at 588 nm and the diminution of the absorbance of free retinal at 340–385 nm. Up to about 30 min of reconstitution, the presence of an isosbestic point at 430 nm indicates that the reaction can be described with two absorbing species—that is, free retinal and the product, the 588-nm pigment. At later times an isosbestic point no longer exists, as apparently additional reactions take place. It was possible to calculate an extinction coefficient for the 588-nm pigment, and a value of 48,000 M –1 cm –1 was obtained assuming that the chromophore contains one retinal per molecule.
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Halorhodopsin — a second retinal pigment in Halobacterium halobium
Trends in Biochemical Sciences, 2003Co-Authors: Janos K. LanyiAbstract:Abstract A light-driven sodium pump has been identified in Halobacterium halobium membranes. It appears to be a retinal protein, with properties similar to those of bacteriorhodopsin. The pigment may play an important role in the presence of this microorganism to illumination.
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characterization of the azide dependent bacteriorhodopsin like photocycle of salinarum Halorhodopsin
Biophysical Journal, 2002Co-Authors: Melinda Lakatos, Janos K. Lanyi, Geza I Groma, Constanta Ganea, Gyorgy VaroAbstract:The photocycle of salinarum Halorhodopsin was investigated in the presence of azide. The azide binds to the Halorhodopsin with 150 mM binding constant in the absence of chloride and with 250 mM binding constant in the presence of 1 M chloride. We demonstrate that the azide-binding site is different from that of chloride, and the influence of chloride on the binding constant is indirect. The analysis of the absorption kinetic signals indicates the existence of two parallel photocycles. One belongs to the 13-cis retinal containing protein and contains a single red shifted intermediate. The other photocycle, of the all-trans retinal containing Halorhodopsin, resembles the cycle of bacteriorhodopsin and contains a long-living M intermediate. With time-resolved spectroscopy, the spectra of intermediates were determined. Intermediates L, N, and O were not detected. The multiexponential rise and decay of the M intermediate could be explained by the introduction of the "spectrally silent" intermediates M1, M2, and HR', HR, respectively. The electric signal measurements revealed the existence of a component equivalent with a proton motion toward the extracellular side of the membrane, which appears during the M1 to M2 transition. The differences between the azide-dependent photocycle of salinarum Halorhodopsin and pharaonis Halorhodopsin are discussed.
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characterization of the proton transporting photocycle of pharaonis Halorhodopsin
Biophysical Journal, 2000Co-Authors: Agnes Kulcsar, Janos K. Lanyi, Geza I Groma, Gyorgy VaroAbstract:The photocycle of pharaonis Halorhodopsin was investigated in the presence of 100 mM NaN(3) and 1 M Na(2)SO(4). Recent observations established that the replacement of the chloride ion with azide transforms the photocycle from a chloride-transporting one into a proton-transporting one. Kinetic analysis proves that the photocycle is very similar to that of bacteriorhodopsin. After K and L, intermediate M appears, which is missing from the chloride-transporting photocycle. In this intermediate the retinal Schiff base deprotonates. The rise of M in Halorhodopsin is in the microsecond range, but occurs later than in bacteriorhodopsin, and its decay is more accentuated multiphasic. Intermediate N cannot be detected, but a large amount of O accumulates. The multiphasic character of the last step of the photocycle could be explained by the existence of a HR' state, as in the chloride photocycle. Upon replacement of chloride ion with azide, the fast electric signal changes its sign from positive to negative, and becomes similar to that detected in bacteriorhodopsin. The photocycle is enthalpy-driven, as is the chloride photocycle of Halorhodopsin. These observations suggest that, while the basic charge translocation steps become identical to those in bacteriorhodopsin, the storage and utilization of energy during the photocycle remains unchanged by exchanging chloride with azide.
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charge motions during the photocycle of pharaonis Halorhodopsin
Biophysical Journal, 2000Co-Authors: Krisztina Ludmann, Janos K. Lanyi, Grazyna Ibron, Gyorgy VaroAbstract:Abstract Oriented gel samples were prepared from Halorhodopsin-containing membranes from Natronobacterium pharaonis, and their photoelectric responses to laser flash excitation were measured at different chloride concentrations. The fast component of the current signal displayed a characteristic dependency on chloride concentration, and could be interpreted as a sum of two signals that correspond to the responses at high-chloride and no-chloride, but high-sulfate, concentration. The chloride concentration-dependent transition between the two signals followed the titration curve determined earlier from spectroscopic titration. The voltage signal was very similar to that reported by another group (Kalaidzidis, I. V., Y. L. Kalaidzidis, and A. D. Kaulen. 1998. FEBS Lett. 427:59–63). The absorption kinetics, measured at four wavelengths, fit the kinetic model we had proposed earlier. The calculated time-dependent concentrations of the intermediates were used to fit the voltage signal. Although no negative electric signal was observed at high chloride concentration, the calculated electrogenicity of the K intermediate was negative, and very similar to that of bacteriorhodopsin. The late photocycle intermediates (O, HR′, and HR) had almost equal electrogenicities, explaining why no chloride-dependent time constant was identified earlier by Kalaidzidis et al. The calculated electrogenicities, and the spectroscopic information for the chloride release and uptake steps of the photocycle, suggest a mechanism for the chloride-translocation process in this pump.
Dieter Oesterhelt - One of the best experts on this subject based on the ideXlab platform.
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Picosecond events in the photochemical cycle of the light-driven chloride-pump Halorhodopsin
Biophysical Journal, 2009Co-Authors: Hans-joachim Polland, Peter Hegemann, Wolfgang Zinth, M. A. Franz, Wolfgang Kaiser, Dieter OesterheltAbstract:The early events in Halorhodopsin after light excitation are studied with picosecond time resolution. Absorption and fluorescence measurements show that the electronically excited state of the incorporated retinal has a lifetime of 5 ps. Within that time a red-shifted photoproduct is formed that remains stable for at least 2 ns.
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The crystal structure of the L1 intermediate of Halorhodopsin at 1.9 angstrom resolution
Photochemistry and Photobiology, 2007Co-Authors: Walter Gmelin, Joachim Heberle, Jorg Tittor, Kornelius Zeth, Ruslan Efremov, Dieter OesterheltAbstract:: The mutant T203V of the light driven chloride pump Halorhodopsin from Halobacterium salinarum was crystallized and the X-ray structure was solved at 1.6 angstroms resolution. The T203V structure turned out to be nearly identical to the wild type protein with a root mean square deviation of 0.43 angstroms for the carbon alpha atoms of the protein backbone. Two chloride binding (CB) sites were demonstrated by a substitution of chloride with bromide and an analysis of anomalous difference Fourier maps. The CB1 site was found at the same position as in the wild type structure. In addition, a second chloride binding site CB2 was identified around Q105 due to higher resolution in the mutant crystal. As T203V showed a 10 times slower decay of its photocycle intermediate L, this intermediate could be trapped with an occupancy of 60% upon illumination at room temperature and subsequent cooling to 120 degrees K. Fourier transform infrared spectroscopy clearly identified the crystal to be trapped in the L1 intermediate state and the X-ray structure was solved to 1.9 angstroms resolution. In this intermediate, the chloride moved by 0.3 angstroms within binding site CB1 as indicated by peaks in difference Fourier density maps. The chloride in the second binding site CB2 remained unchanged. Thus, intraproteinous chloride translocation from the extracellular to the cytoplasmic part of the protein must occur in reaction steps following the L1 intermediate in the catalytic cycle of Halorhodopsin.
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structure of the light driven chloride pump Halorhodopsin at 1 8 a resolution
Science, 2000Co-Authors: Michael Kolbe, Huseyin Besir, Larsoliver Essen, Dieter OesterheltAbstract:Halorhodopsin, an archaeal rhodopsin ubiquitous in Haloarchaea, uses light energy to pump chloride through biological membranes. Halorhodopsin crystals were grown in a cubic lipidic phase, which allowed the x-ray structure determination of this anion pump at 1.8 angstrom resolution. Halorhodopsin assembles to trimers around a central patch consisting of palmitic acid. Next to the protonated Schiff base between Lys 242 and the isomerizable retinal chromophore, a single chloride ion occupies the transport site. Energetic calculations on chloride binding reveal a combination of ion-ion and ion-dipole interactions for stabilizing the anion 18 angstroms below the membrane surface. Ion dragging across the protonated Schiff base explains why chloride and proton translocation modes are mechanistically equivalent in archaeal rhodopsins.
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the three dimensional structure of Halorhodopsin to 5 a by electron crystallography a new unbending procedure for two dimensional crystals by using a global reference structure
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Edmund R S Kunji, Dieter Oesterhelt, Susanne Von Gronau, Richard HendersonAbstract:Electron microscopy does not, in principle, require highly ordered crystals to determine a high-resolution structure. Nevertheless, crystals of any type help to constrain the molecules into a more limited range of orientations and positions, from which it is easier to carry out structure determination. We describe an improved procedure for determination of crystalline disorder, which we have applied to poorly ordered two-dimensional crystals of the chloride pump Halorhodopsin from Halobacterium salinarum. The new image analysis procedure involves the use of a reference projection calculated from a global three-dimensional map to carry out the initial cross-correlation analysis. Coupled with a greater number of images taken with field emission gun microscopes, this has allowed us to calculate a three-dimensional structure for Halorhodopsin, in which the seven transmembrane helices and certain molecular features, such as the β-ionone ring of retinal, are now resolved.
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the initial reaction dynamics of the light driven chloride pump Halorhodopsin
Chemical Physics Letters, 1995Co-Authors: T Arlt, Stefan Schmidt, Wolfgang Zinth, Ulrich Haupts, Dieter OesterheltAbstract:Abstract The light-induced dynamics of Halorhodopsin has been studied by transient absorption spectroscopy with a time resolution better than 100 fs. Similar to the process known from bacteriorhodopsin a fast initial process with a time constant of 170 ± 70 fs occurs indicating motions of the retinal chromophore on the excited-state potential energy surface. The decay of the excited electronic state is biexponential with time constants of 1.5 ± 0.7 and 8.5 ± 1.5 ps. The formation of the first red-shifted ground-state photoproduct occurs on the same timescale. A qualitative model for the primary reactions is proposed, which explains the lower quantum yield Φ of about 0.3 in Halorhodopsin compared to 0.6 in bacteriorhodopsin.
Martin Engelhard - One of the best experts on this subject based on the ideXlab platform.
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Microbial Halorhodopsins: Light-Driven Chloride Pumps
Chemical Reviews, 2018Co-Authors: Christopher Engelhard, Igor Chizhov, Friedrich Siebert, Martin EngelhardAbstract:Early research on the four microbial rhodopsins discovered in the archaeal Halobacterium salinarum revealed a structural template that served as a scaffold for two different functions: light-driven ion transport and phototaxis. Bacteriorhodopsin and Halorhodopsin are proton and chloride pumps, respectively, while sensory rhodopsin I and II are responsible for phototactic behavior of the archaea. Halorhodopsins have been identified in various other species. Besides this group of archaeal Halorhodopsins distinct chloride transporting rhodopsins groups have recently been identified in other organism like Flavobacteria or Cyanobacteria. Halorhodopsin from Natronomonas pharaonis is the best-studied homologue because of its facile expression and purification and its advantageous properties, which was the reason to introduce this protein as neural silencer into the new field of optogenetics. Two other major families of genetically encoded silencing proteins, proton pumps and anion channels, extended the repertoi...
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anion uptake in Halorhodopsin from natromonas pharaonis studied by ftir spectroscopy consequences for the anion transport mechanism
Biochemistry, 2006Co-Authors: Jarmila Guijarro, Martin Engelhard, Friedrich SiebertAbstract:The uptake of chloride, bromide, iodide, nitrate, and azide by anion-depleted blue Halorhodopsin from Natronobacterium pharaonis has been followed by FTIR difference spectroscopy using an ATR sampling device. The spectra are compared with the spectrum of the O intermediate obtained by time-resolved FTIR studies of the photocycle. It is demonstrated that anion-free blue Halorhodopsin can be identified with the O intermediate and, thus, that the decay of O is due to the passive uptake of the anion. The great similarity of the anion-binding spectra and their identity in the case of the monoatomic anions indicate a rather unspecific binding site for the different anions dominated by electrostatic interactions. Comparing spectra obtained with 15N nitrate and unlabeled nitrate, the NO-stretching bands could be identified. The small splitting and the small IR intensity of those bands indicate a rather nonpolar binding site with a rather isotropic influence on the nitrate, in contrast to aqueous nitrate. In furth...
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Effect of anions on the photocycle of Halorhodopsin. Substitution of chloride with formate anion.
Biochemistry, 2005Co-Authors: Keren Mevorat-kaplan, Martin Engelhard, Vlad Brumfeld, Mordechai ShevesAbstract: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 si...
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purification of histidine tagged bacteriorhodopsin pharaonis Halorhodopsin and pharaonis sensory rhodopsin ii functionally expressed in escherichia coli
FEBS Letters, 1999Co-Authors: Ilja P Hohenfeld, Ansgar A Wegener, Martin EngelhardAbstract:Bacteriorhodopsin (BR) from Halobacterium salinarum as well as Halorhodopsin (pHR) and sensory rhodopsin II (pSRII) from Natronobacterium pharaonis were functionally expressed in E. coli using the method of Shimono et al. [FEBS Lett. (1997) 420, 54–56]. The histidine tagged proteins were purified with yields up to 1.0 mg/l cell culture and characterized by ESI mass spectrometry and their photocycle. The pSRII and pHR photocycles were indistinguishable from the wild type proteins. The BR photocycle was considerably prolonged. pSOII is located in the cytoplasmic membrane and the C-terminus is oriented towards the cytoplasm as determined by immunogold labelling.
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chromophore anion interactions in Halorhodopsin from natronobacterium pharaonis probed by time resolved resonance raman spectroscopy
Biochemistry, 1997Co-Authors: Sandra Gerscher, Muthusamy Mylrajan, Peter Hildebrandt, Mariehelene Baron, Renate Muller, Martin EngelhardAbstract: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 CC and CN 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 CC and CN ...
Gyorgy Varo - One of the best experts on this subject based on the ideXlab platform.
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characterization of the azide dependent bacteriorhodopsin like photocycle of salinarum Halorhodopsin
Biophysical Journal, 2002Co-Authors: Melinda Lakatos, Janos K. Lanyi, Geza I Groma, Constanta Ganea, Gyorgy VaroAbstract:The photocycle of salinarum Halorhodopsin was investigated in the presence of azide. The azide binds to the Halorhodopsin with 150 mM binding constant in the absence of chloride and with 250 mM binding constant in the presence of 1 M chloride. We demonstrate that the azide-binding site is different from that of chloride, and the influence of chloride on the binding constant is indirect. The analysis of the absorption kinetic signals indicates the existence of two parallel photocycles. One belongs to the 13-cis retinal containing protein and contains a single red shifted intermediate. The other photocycle, of the all-trans retinal containing Halorhodopsin, resembles the cycle of bacteriorhodopsin and contains a long-living M intermediate. With time-resolved spectroscopy, the spectra of intermediates were determined. Intermediates L, N, and O were not detected. The multiexponential rise and decay of the M intermediate could be explained by the introduction of the "spectrally silent" intermediates M1, M2, and HR', HR, respectively. The electric signal measurements revealed the existence of a component equivalent with a proton motion toward the extracellular side of the membrane, which appears during the M1 to M2 transition. The differences between the azide-dependent photocycle of salinarum Halorhodopsin and pharaonis Halorhodopsin are discussed.
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characterization of the proton transporting photocycle of pharaonis Halorhodopsin
Biophysical Journal, 2000Co-Authors: Agnes Kulcsar, Janos K. Lanyi, Geza I Groma, Gyorgy VaroAbstract:The photocycle of pharaonis Halorhodopsin was investigated in the presence of 100 mM NaN(3) and 1 M Na(2)SO(4). Recent observations established that the replacement of the chloride ion with azide transforms the photocycle from a chloride-transporting one into a proton-transporting one. Kinetic analysis proves that the photocycle is very similar to that of bacteriorhodopsin. After K and L, intermediate M appears, which is missing from the chloride-transporting photocycle. In this intermediate the retinal Schiff base deprotonates. The rise of M in Halorhodopsin is in the microsecond range, but occurs later than in bacteriorhodopsin, and its decay is more accentuated multiphasic. Intermediate N cannot be detected, but a large amount of O accumulates. The multiphasic character of the last step of the photocycle could be explained by the existence of a HR' state, as in the chloride photocycle. Upon replacement of chloride ion with azide, the fast electric signal changes its sign from positive to negative, and becomes similar to that detected in bacteriorhodopsin. The photocycle is enthalpy-driven, as is the chloride photocycle of Halorhodopsin. These observations suggest that, while the basic charge translocation steps become identical to those in bacteriorhodopsin, the storage and utilization of energy during the photocycle remains unchanged by exchanging chloride with azide.
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analogies between Halorhodopsin and bacteriorhodopsin
Biochimica et Biophysica Acta, 2000Co-Authors: Gyorgy VaroAbstract:The light-activated proton-pumping bacteriorhodopsin and chloride ion-pumping Halorhodopsin are compared. They belong to the family of retinal proteins, with 25% amino acid sequence homology. Both proteins have seven α helices across the membrane, surrounding the retinal binding pocket. Photoexcitation of all-trans retinal leads to ion transporting photocycles, which exhibit great similarities in the two proteins, despite the differences in the ion transported. The spectra of the K, L, N and O intermediates, calculated using time-resolved spectroscopic measurements, are very similar in both proteins. The absorption kinetic measurements reveal that the chloride ion transporting photocycle of Halorhodopsin does not have intermediate M characteristic for deprotonated Schiff base, and intermediate L dominates the process. Energetically the photocycle of bacteriorhodopsin is driven mostly by the decrease of the entropic energy, while the photocycle of Halorhodopsin is enthalpy-driven. The ion transporting steps were characterized by the electrogenicity of the intermediates, calculated from the photoinduced transient electric signal measurements. The function of both proteins could be described with the ‘local access’ model developed for bacteriorhodopsin. In the framework of this model it is easy to understand how bacteriorhodopsin can be converted into a chloride pump, and Halorhodopsin into a proton pump, by changing the ion specificity with added ions or site-directed mutagenesis.
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charge motions during the photocycle of pharaonis Halorhodopsin
Biophysical Journal, 2000Co-Authors: Krisztina Ludmann, Janos K. Lanyi, Grazyna Ibron, Gyorgy VaroAbstract:Abstract Oriented gel samples were prepared from Halorhodopsin-containing membranes from Natronobacterium pharaonis, and their photoelectric responses to laser flash excitation were measured at different chloride concentrations. The fast component of the current signal displayed a characteristic dependency on chloride concentration, and could be interpreted as a sum of two signals that correspond to the responses at high-chloride and no-chloride, but high-sulfate, concentration. The chloride concentration-dependent transition between the two signals followed the titration curve determined earlier from spectroscopic titration. The voltage signal was very similar to that reported by another group (Kalaidzidis, I. V., Y. L. Kalaidzidis, and A. D. Kaulen. 1998. FEBS Lett. 427:59–63). The absorption kinetics, measured at four wavelengths, fit the kinetic model we had proposed earlier. The calculated time-dependent concentrations of the intermediates were used to fit the voltage signal. Although no negative electric signal was observed at high chloride concentration, the calculated electrogenicity of the K intermediate was negative, and very similar to that of bacteriorhodopsin. The late photocycle intermediates (O, HR′, and HR) had almost equal electrogenicities, explaining why no chloride-dependent time constant was identified earlier by Kalaidzidis et al. The calculated electrogenicities, and the spectroscopic information for the chloride release and uptake steps of the photocycle, suggest a mechanism for the chloride-translocation process in this pump.
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proton transport by Halorhodopsin
Biochemistry, 1996Co-Authors: Gyorgy Varo, Richard Needleman, Leonid S Brown, Janos K. LanyiAbstract:In Halorhodopsin from Natronobacterium pharaonis, a light-driven chloride pump, the chloride binding site also binds azide. When azide is bound at this location the retinal Schiff base transiently ...
Laszlo Zimanyi - One of the best experts on this subject based on the ideXlab platform.
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fourier transform raman study of retinal isomeric composition and equilibration in Halorhodopsin
Journal of Physical Chemistry B, 1997Co-Authors: Laszlo Zimanyi, Janos K. LanyiAbstract:Earlier results suggested that the description of the photoreaction of Halorhodopsin depends on our being able to distinguish and dissect the photoproducts of the all-trans chromophore from those of the 13-cis,15-syn chromophore. We have used FT Raman spectroscopy, a nonperturbing method, to analyze the isomeric states of the retinal under various conditions in Halorhodopsins from Halobacterium salinarium and Natronobacterium pharaonis. The results indicate that light adaptation occurs in the former protein, and the amounts of the two isomers in the light-adapted and dark-adapted forms are consistent with the weights given to them, from spectroscopic and mathematical criteria, in the earlier calculations. No light adaptation occurs in the latter protein, which contains mostly, but not entirely, all-trans-retinal. There is no light adaptation in either protein in the absence of chloride, the transported substrate of these anion pumps, and in spite of a 13-cis-like photocycle, they contain both all-trans- a...
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photocycle of Halorhodopsin from halobacterium salinarium
Biophysical Journal, 1995Co-Authors: Gyorgy Varo, Laszlo Zimanyi, Richard Needleman, Janos K. LanyiAbstract:The light-driven chloride pump, Halorhodopsin, is a mixture containing all-trans and 13-cis retinal chromophores under both light and dark-adapted conditions and can exist in chloride-free and chloride-binding forms. To describe the photochemical cycle of the all-trans, chloride-binding state that is associated with the transport, and thereby initiate study of the chloride translocation mechanism, one must first dissect the contributions of these species to the measured spectral changes. We resolved the multiple photochemical reactions by determining flash-induced difference spectra and photocycle kinetics in Halorhodopsin-containing membranes prepared from Halobacterium salinarium, with light- and dark-adapted samples at various chloride concentrations. The high expression of cloned Halorhodopsin made it possible to do these measurements with unfractionated cell envelope membranes in which the chromophore is photostable not only in the presence of NaCl but also in the Na2SO4 solution used for reference. Careful examination of the flash-induced changes at selected wavelengths allowed separating the spectral changes into components and assigning them to the individual photocycles. According to the results, a substantial revision of the photocycle model for H. salinarium Halorhodopsin, and its dependence on chloride, is required. The cycle of the all-trans chloride-binding form is described by the scheme, HR-hv-->K L1 L2 N-->HR, where HR, K, L, and N designate Halorhodopsin and its photointermediates. Unlike the earlier models, this is very similar to the photoreaction of bacteriorhodopsin when deprotonation of the Schiff base is prevented (e.g., at low pH or in the D85N mutant). Also unlike in the earlier models, no step in this photocycle was noticeably affected when the chloride concentration was varied between 20 mM and 2 M in an attempt to identify a chloride-binding reaction.
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Anion binding to the chloride pump, Halorhodopsin, and its implications for the transport mechanism
FEBS Letters, 1990Co-Authors: Janos K. Lanyi, Gyorgy Varo, Albert Duschi, Laszlo ZimanyiAbstract: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.
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properties and photochemistry of a Halorhodopsin from the haloalkalophile natronobacterium pharaonis
Journal of Biological Chemistry, 1990Co-Authors: Albert Duschl, Janos K. Lanyi, Laszlo ZimanyiAbstract:Abstract Pharaonis Halorhodopsin is a light-driven transport system for chloride, similarly to the previously described Halorhodopsin, but we find that it transports nitrate as effectively as chloride. We studied the photoreactions of the purified, detergent-solubilized pharaonis pigment with a gated multichannel analyzer. At a physiological salt concentration (4 M NaCl), the absorption spectra and rate constants of rise and decay for intermediates of the photocycle were similar to those for Halorhodopsin. In buffer containing nitrate, Halorhodopsin exhibits a second, truncated photocycle; this difference in the photoreaction of the pigment occurs when an anion is bound in such a way as to preclude transport. As expected from the lack of anion specificity in the transport, the photocycle of pharaonis Halorhodopsin was nearly unaffected by replacement of chloride with nitrate. All presumed buried positively charged residues, which might play a role in anion binding, are conserved in the two pigments. At the extracellular end of the presumed helix C, however, an arginine residue is found in Halorhodopsin, but not in pharaonis Halorhodopsin, and an arginine-rich segment between the presumed helices A and B in Halorhodopsin is replaced by a less positively charged sequence in pharaonis Halorhodopsin (Lanyi, J. K., Duschl, A., Hatfield, G. W., May, K., and Oesterhelt, D. (1990) J. Biol. Chem. 265, 1253-1260). One or both of these alterations may explain the difference in the anion selectivity of the two proteins.
