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PubMed2001

Light-induced structural changes occur in the transmembrane helices of the Natronobacterium pharaonis HtrII transducer.

The Natronobacterium pharaonis HtrII (NpHtrII) transducer interacts with its cognate photoactive sensory rhodopsin receptor, NpSRII, to mediate phototaxis responses. NpHtrII is predicted to have two transmembrane helices and a large cytoplasmic domain and to form a homodimer. Single cysteines were substituted into an engineered cysteine-less NpHtrII at 38 positions in its transmembrane domain. Oxidative disulfide cross-linking efficiencies of the monocysteine mutants were measured with or without photoactivation of NpSRII. The rapid cross-linking rates at several positions support that NpHtrII is a dimer when functionally expressed in the Halobacterium salinarum membrane. Thirteen positions in the second transmembrane segment (TM2) exhibited significant light-induced increases in cross-linking efficiency, and they define a single face traversing the length of the segment when modeled as an alpha-helix. Four positions in this helix showing light-induced decreases in efficiency are clustered on the cytoplasmic side of the protein. One of the monocysteine mutants, G83C, showed loss of phototaxis responses, and analysis of double mutants showed that the G83C mutation alters the dark structure of the TM2-TM2' region of NpHtrII. In summary, the results reveal conformationally active regions in the second transmembrane segment of NpHtrII and a face along the length of TM2 that becomes more available for TM2-TM2' cross-linking upon receptor photoactivation. The data also establish that one residue in TM2, Gly83, is critical for maintaining the proper conformation of NpHtrII for signal relay from the photoactivated receptor to the kinase-binding region of the transducer.

Amino Acid Sequence↗
PubMed2002

Stopped-flow analysis on anion binding to blue-form halorhodopsin from Natronobacterium pharaonis: comparison with the anion-uptake process during the photocycle.

Pharaonis halorhodopsin (phR), the light-driven chloride ion pump from Natronobacterium pharaonis with C-terminal histidine tag, was expressed in Escherichia coli cells. The protein was solubilized with 0.1% n-dodecyl beta-D-maltopyranoside and purified with a nickel column. Removal of Cl- from the medium yields blue phR (phR(blue)) that has lost Cl- near the chromophore. Addition of Cl- converts phR(blue) to a red-shifted Cl--bound form (phR(Cl)). Circular dichroic spectra of phR(blue) and phR(Cl) exhibited a bilobe in the visual region, indicating specific oligomerization of the phR monomers. The order of anion concentration which induced a shift from phR(blue) to phR(X) was Br- < Cl- < NO3- < N3-, which was the same as in the case of phR purified from N. pharaonis membranes. Chloride binding kinetics was measured by time-resolved absorption changes with stopped-flow rapid mixing. Rates of Cl- binding consisted of fast and slow components, and the amplitude of the fast component was about 90% of the total changes. The rate constant of the fast component at 100 mM NaCl at 25 degrees C was 260 s(-1) with an apparent activation energy of 35 kJ/mol. These values are in good agreement with the process of Cl- uptake in the photocycle (O --> hR' reaction) reported previously [Váró et al. (1995) Biochemistry 34, 14500-14507]. In addition, the Cl- concentration dependence on both rates was similar to each other. These observations suggest that the O-intermediate is similar to phR(blue) and that Cl- uptake during the photocycle may be ruled by a passive process.

Anions↗
PubMed2004

Consequences of counterion mutation in sensory rhodopsin II of Natronobacterium pharaonis for photoreaction and receptor activation: an FTIR study.

In many retinal proteins the proton transfer from the Schiff base to the counterion represents a functionally important step of the photoreaction. In the signaling state of sensory rhodopsin II from Natronobacterium pharaonis this transfer has already occurred, but in the counterion mutant Asp75Asn it is blocked during all steps of the photocycle. Therefore, the study of the molecular changes during the photoreaction of this mutant should provide a deeper understanding of the activation mechanism, and for this, we have applied time-resolved step-scan FTIR spectroscopy. The photoreaction is drastically altered; only red-shifted intermediates are formed with a chromophore strongly twisted around the 14-15 single bond. In addition, the photocycle is shortened by 2 orders of magnitude. Nevertheless, a transition involving only protein changes similar to that of the wild type is observed, which has been correlated with the formation of the signaling state. However, whereas in the wild type this transition occurs in the millisecond range, it is shortened to 200 micros in the mutant. The results are discussed with respect to the altered electrostatic interactions, role of proton transfer, the published 3D structure, and physiological activity.

Archaeal Proteins↗
PubMed2005

Hydrogen-bonding alterations of the protonated Schiff base and water molecule in the chloride pump of Natronobacterium pharaonis.

Halorhodopsin is a light-driven chloride ion pump. Chloride ion is bound in the Schiff base region of the retinal chromophore, and unidirectional chloride transport is probably enforced by the specific hydrogen-bonding interaction with the protonated Schiff base and internal water molecules. In this article, we study hydrogen-bonding alterations of the Schiff base and water molecules in halorhodopsin of Natronobacterium pharaonis (pHR) by assigning their N-D and O-D stretching vibrations in D(2)O, respectively. Highly accurate low-temperature Fourier transform infrared spectroscopy revealed that hydrogen bonds of the Schiff base and water molecules are weak in the unphotolyzed state, whereas they are strengthened upon retinal photoisomerization. Halide dependence of the stretching vibrations enabled us to conclude that the Schiff base forms a direct hydrogen bond with Cl(-) only in the K intermediate. Hydrogen bond of the Schiff base is further strengthened in the L(1) intermediate, whereas the halide dependence revealed that the acceptor is not Cl(-), but presumably a water molecule. Thus, it is concluded that the hydrogen-bonding interaction between the Schiff base and Cl(-) is not a driving force of the motion of Cl(-). Rather, the removal of its hydrogen bonds with the Schiff base and water(s) makes the environment around Cl(-) less polar in the L(1) intermediate, which presumably drives the motion of Cl(-) from its binding site to the cytoplasmic domain.

Chlorides↗
PubMed2006

Shape and oligomerization state of the cytoplasmic domain of the phototaxis transducer II from Natronobacterium pharaonis.

Phototaxis allows archaea to adjust flagellar motion in response to light. In the photophobic response of Natronobacterium pharaonis, light-activated sensory rhodopsin II causes conformational changes in the transducer II protein (pHtrII), initiating the two-component signaling system analogous to bacterial chemotaxis. pHtrII's cytoplasmic domain (pHtrII-cyt) is homologous to the cytoplasmic domains of eubacterial chemotaxis receptors. Chemotaxis receptors require dimerization for activity and are in vivo-organized in large clusters. In this study we investigated the oligomerization and aggregation states of pHtrII-cyt by using chemical cross-linking, analytical gel-filtration chromatography, and small-angle neutron scattering. We show that pHtrII-cyt is monomeric in dilute buffers, but forms dimers in 4 M KCl, the physiological salt concentration for halophilic archaea. At high ammonium sulfate concentration, the protein forms higher-order aggregates. The monomeric protein has a rod-like shape, 202 A in length and 14.4 A in diameter; upon dimerization the length increases to 248 A and the diameter to 18.2 A. These results suggest that under high salt concentration the shape and oligomerization state of pHtrII-cyt are comparable to those of chemotaxis receptors.

Archaeal Proteins↗
PubMed2003

Importance of the broad regional interaction for spectral tuning in Natronobacterium pharaonis phoborhodopsin (sensory rhodopsin II).

Natronobacterium pharaonis phoborhodopsin (ppR; also called N. pharaonis sensory rhodopsin II, NpsRII) is a photophobic sensor in N. pharaonis, and has a shorter absorption maximum (lambdamax, 500 nm) than those of other archaeal retinal proteins (lambdamax, 560-590 nm) such as bacteriorhodopsin (bR). We constructed chimeric proteins between bR and ppR to investigate the long range interactions effecting the color regulation among archaeal retinal proteins. The lambdamax of B-DEFG/P-ABC was 545 nm, similar to that of bR expressed in Escherichia coli (lambdamax, 550 nm). B-DEFG/P-ABC means a chimera composed of helices D, E, F, and G of bR and helices A, B, and C of ppR. This indicates that the major factor(s) determining the difference in lambdamax between bR and ppR exist in helices DEFG. To specify the more minute regions for the color determination between bR and ppR, we constructed 15 chimeric proteins containing helices D, E, F, and G of bR. According to the absorption spectra of the various chimeric proteins, the interaction between helices D and E as well as the effect of the hydroxyl group around protonated Schiff base on helix G (Thr-204 for ppR and Ala-215 for bR) are the main factors for spectral tuning between bR and ppR.

Amino Acid Sequence↗
PubMed2001

Thermodynamics of the early steps in the photocycle of Natronobacterium pharaonis halorhodopsin. Influence of medium and of anion substitution.

The enthalpy (delta H) and structural volume changes (delta V) associated with the formation and decay of the early intermediate K600 in the photocycle of Natronobacterium pharaonis halorhodopsin (pHR), an inward-directed anion pump, were obtained by laser-induced optoacoustic spectroscopy. A large expansion is associated with K600 formation, its value depending on the medium and on the anion (Cl-, NO3-, Br-, I-). A smaller expansion is associated with K600 decay to L520. A contraction is found for the same step in the case of the azide-loaded pHR which is an efficient outward-directed proton pump. Thus, the conformational changes in L520 determine the direction and sign of charge translocation. The linear correlation between delta H and delta V for chloride-loaded pHR observed upon mild medium variations is attributed to enthalpy-entropy compensation effects and allows the calculation of the free-energy changes, delta GK = (97 +/- 16) kJ/mol and delta GKL = -(2 +/- 2) kJ/mol. Different from other systems, delta S correlates negatively with delta V in the first steps of the pHR photocycle. Thus, the space around the anion becomes larger and more rigid during each of these two steps. The photocycle quantum yield was 0.52 for chloride-pHR as measured by laser flash photolysis.

Anions↗
PubMed2003

Interaction of Natronobacterium pharaonis phoborhodopsin (sensory rhodopsin II) with its cognate transducer probed by increase in the thermal stability.

Pharaonis phoborhodopsin (ppR, also called Natronobacterium pharaonis sensory rhodopsin II) and its transducer protein, pharaonis halobacterial transducer of ppR (pHtrII), form a signaling complex, and light signals are transmitted from the sensor to the transducer by the protein-protein interaction. A truncated pHtrII(1-159) consisting of intramembrane helices (expressing amino acid residues from the first to the 159th position) and ppR form the complex in a solution containing 0.1% n-dodecyl-beta-D-maltoside. At 75-85 degrees C, the time-dependent color loss of ppR was caused by denaturation. We found that pHtrII(1-159) retarded the denaturation rate of ppR. This increase in the thermal stability was used as a probe for the binding ability in the dark. Tyr199 of ppR and Asn74 of pHtrII(1-114) were proposed as amino acid residues interacting with each other through hydrogen bonding. Then,ppR and pHtrII(1-159) mutants at these positions were prepared to examine the effect on the binding in the dark. The wild-type and Y199F mutant can bind pHtrII(1-159), suggesting that the hydrogen bonding between these specific amino acid residues may not be the only cause of the binding, but the hydrophobic interaction via phenyl ring of ppR may contribute dominantly.

Archaeal Proteins↗
PubMedDate not supplied

The protonated Schiff base of halorhodopsin from Natronobacterium pharaonis is hydrolyzed at elevated temperatures.

Halorhodopsin from Natronobacterium pharaonis (pHR) is a light-driven chloride pump in which photoisomerzation of a retinal chromophore triggers a photocycle which leads to a chloride anion transport across the plasma membrane. Similarly to other retinal proteins the protonated Schiff base (PSB), which covalently links the retinal to the protein, does not experience hydrolysis reaction at room temperature even though several water molecules are located in the protonated Schiff base (PSB) vicinity. In the present studies we have revealed that in contrast to other studied archaeal rhodopsins, temperature increase to about 70 degrees C hydrolyses the PSB linkage of pHR. The rate of the reaction is affected by Cl-concentration and reveals an anion binding site (in addition to the Cl- in the SB vicinity) with a binding constant of 100mM (measured at 70 degrees C). We suggest that this binding site is located on the extracellular side and its possible role in the Cl-pumping mechanism is discussed. The rate of the hydrolysis reaction is affected by the nature of the anion bound to pHR. Substitution of the Cl- anion by Br-, I- and SCN- exhibits similar behavior to that of CI- in the region of 100mM but higher concentrations are needed for N3-, HCOO- and NO2-to achieve similar behavior. Steady state pigment illumination accelerates the reaction and reduces the energy of activation and the frequency factor. Adjusting the sample temperature to 25 degrees C following the hydrolysis reaction led to about 80% pigment recovery. However, the newly reformed pigment is different from the mother pigment and has different characteristics. It is concluded that the apo-membrane adopts a modified conformation and/or aggregated state which rebinds the retinal to give a new conformation of the pHR pigment.

Archaeal Proteins↗
PubMed1997

The characterization of the nv-gvpACNOFGH gene cluster involved in gas vesicle formation in Natronobacterium vacuolatum.

The haloalkaliphilic archaeon Natronobacterium vacuolatum forms cylinder-shaped gas vesicles throughout the growth cycle when grown in media containing 15-25% NaCl. Cells cultivated in media containing 13% NaCl are, however, gas-vesicle-free. The major gas vesicle structural protein, nv-GvpA, was detected by an antiserum raised against the gas vesicles of Haloferax mediterranei; the antiserum reacted with an 8.3-kDa protein in samples containing cell extracts or purified gas vesicles of N. vacuolatum. The gene encoding nv-GvpA was isolated together with six additional gvp genes; these genes are arranged consecutively in a cluster as nv-gvpACNOFGH and are cotranscribed. Transcript analysis by primer extension revealed only one start site three nucleotides upstream of the nv-gvpA reading frame. This arrangement of gvp genes differs from that of the gas-vesicle-encoding genes in Halobacterium salinarium and Hf. mediterranei. The comparison of the deduced Gvp protein sequences indicated similarities with the respective halobacterial Gvp proteins, with GvpA exhibiting the highest degree of conservation (97-100%). The second gas vesicle structural protein, nv-GvpC, was 150-250 amino acids longer than all other halobacterial GvpC proteins and was much less conserved (48-73%). The expression of the nv-gvp genes was monitored in N. vacuolatum cells cultivated in 20 or 13% salt media. Northern and Western analyses showed that despite the lack of gas vesicles in cells grown in 13% salt medium, the gvpACNOFGH gene cluster was transcribed and GvpA protein was synthesized, suggesting that the absence of gas vesicles is not due to a lack of transcription.

Amino Acid Sequence↗
PubMed1995

Isolation of photochemically active archaebacterial photoreceptor, pharaonis phoborhodopsin from Natronobacterium pharaonis.

A photoreceptor, pharaonis phoborhodopsin for the negative phototaxis of extremely halophilic and alkalophilic archaebacterium, Natronobacterium pharaonis was isolated in a photochemically active state. A detailed examination of the chromatographic separation made it possible to separate contaminating proteins, such as cytochromes. The procedure resulted in a 2938-fold enrichment with a yield of 15.5%. The isolated pharaonis phoborhodopsin had an absorption maximum at 498 nm, an A280/A498 ratio of 1.27 and a single band near 24 kDa on the SDS-polyacrylamide gels. The isolated pharaonis phoborhodopsin underwent a photochemical reaction after flash excitation. The photocyclic reaction closely resembled that of the membrane-bound pharaonis phoborhodopsin.

Archaea↗
PubMed1994

Phototactic behaviour of the archaebacterial Natronobacterium pharaonis.

Natronobacterium pharaonis can react tactically to photo- and chemostimuli. It moves by rotation of a flagellar bundle which is monopolarly inserted. Under sufficient oxygen supply the photophobic response of N. pharaonis has been measured. The resulting action spectrum matches the absorption spectrum of the purified retinylidene protein psR-II. Retical synthesis could be inhibited by nicotine. Cells grown in the presence of nicotine show a strongly reduced photoresponse, which could be restored by addition of retinal. These data identify psR-II as the receptor for negative phototaxis.

Chemotaxis↗
PubMed1994

A novel prenyltransferase, farnesylgeranyl diphosphate synthase, from the haloalkaliphilic archaeon, Natronobacterium pharaonis.

A novel prenyltransferase, farnesylgeranyl diphosphate (FGPP) synthase (EC 2.5.1.X), which synthesizes C25-prenyl diphosphate, was found in the haloalkaliphilic archaeon Natronobacterium pharaonis. It was separated from geranylgeranyl diphosphate (GGPP) synthase (EC 2.5.1.29), which synthesizes C20-prenyl diphosphate, a major prenyltransferase in this organism. The highest activity of FGPP synthase was observed when GGPP was used as the allylic substrate. FGPP synthase may synthesize a precursor for the C25 moiety of C20, C25 diether lipids using a longer allylic diphosphate, such as GGPP synthesized by GGPP synthase, rather than dimethylallyl diphosphate, which is the product of isopentenyl diphosphate isomerase.

Alkyl and Aryl Transferases↗
PubMed1996

Protonation changes during the photocycle of sensory rhodopsin II from Natronobacterium pharaonis.

The Fourier Transform Infrared (FTIR) spectra of photocycle intermediates of sensory rhodopsin II (pSRII) from Natronobacterium pharaonis were measured. The results of the FTIR experiments indicate considerable conformational movements of pSRII already at the stage of the early K-like intermediate which persist at least during the lifetime of the long lived intermediate. These changes in the amide bond region are more intense than those described for sensory rhodopsin I (SRI) and are quite similar to those observed for rhodopsin. Concomitantly with the deprotonation of the Schiff base a carboxyl group located in a hydrophobic environment is protonated. In analogy to bacteriorhodopsin, this carboxyl group might arise from Asp-75 which probably serves as counter ion to the Schiff base. The protonation reaction differs from the situation observed in SRI where the protonation is pH independent over the range of pH 5-8.

Archaea↗
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↗
PubMed1994

Raman spectroscopic study of the blue copper protein halocyanin from Natronobacterium pharaonis.

Halocyanin, a blue copper protein from Natronobacterium pharaonis was studied by Raman spectroscopic techniques. Near-infrared Fourier transform Raman spectra, which for the first time have been employed to study copper proteins in both oxidation states, display the Raman bands of the protein and, in the oxidized state, also the preresonance-enhanced bands of the copper center. The frequency of the amide I band at 1676 cm-1 indicates a predominant beta-sheet protein structure, which is typical for small blue copper proteins. The Raman spectra provide no evidence for major redox-linked changes of the secondary structure. Resonance Raman spectra of the oxidized protein obtained upon excitation close to the 600-nm absorption band were measured in the pH range between 7.7 and 4.5. The vibrational band pattern in the Cu-cysteine stretching region is closely related to that of azurin, indicating far-reaching similarities of the coordination geometry of the copper center in both proteins. Significantly lower frequencies, however, are noted for the Cu-histidine stretches, which appear as a closely spaced doublet at ca. 260 cm-1. Lowering the pH to 4.5 leads to an increase of this band splitting with one component shifting down to 247 cm-1. This downshift is attributed to the rupture of a hydrogen bond between one of the histidine ligands and a nearby carboxyl group, which becomes protonated at such a low pH. On the other hand, no major changes in the Cu-cysteine stretching region are noted at pH 4.5, implying that the coordination geometry remains largely unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Azurin↗
PubMed1993

Halocyanin, an archaebacterial blue copper protein (type I) from Natronobacterium pharaonis.

A small blue copper protein with a molecular mass of about 15.5 kDa has been isolated from the haloalkaliphilic Natronobacterium pharaonis. This protein, which was named halocyanin, contains one Cu2+. The circular dichroism spectrum in the region of the absorption of the peptide bond reveals mainly beta-structural elements. The absorption spectrum in the visible range has three maxima with the main band at 600 nm. The circular dichroism spectrum in this region is characterized by four extrema with maxima at 413 and 590 nm and minima at 477 nm as well as 721 nm. The electron paramagnetic resonance spectrum displays a hyperfine structure which shows a close resemblance to those of plastocyanin. The cumulative spectral data agree well with those of other small blue copper proteins with axial symmetry of the Cu ligands. In analogy, one can deduce that halocyanin may adopt a type I copper binding site with two His, one Met, and one Cys as probable ligands. This conclusion is confirmed by the C-terminal sequence which contains, homologous to other known sequences of type I copper proteins, three of the four copper ligands. Halocyanin can only be removed from the membrane by mild treatment with detergents. This observation indicates that halocyanin is a peripheral membrane protein which serves as a mobile electron carrier. The presence of type I blue copper proteins is archaebacteria might lead to further insights into their phylogenetic origin.

Amino Acid Sequence↗
PubMed1997

Electron transfer proteins from the haloalkaliphilic archaeon Natronobacterium pharaonis: possible components of the respiratory chain include cytochrome bc and a terminal oxidase cytochrome ba3.

Natronobacterium pharaonis, an aerobic haloalkaliphilic archaebacterium, expresses high concentrations of redox proteins as do alkaliphilic eubacteria. The first redox protein characterized from N. pharaonis was halocyanin [Scharf, B., & Engelhard, M. (1993) Biochemistry 32, 12894-12900], a small blue copper protein. It is a peripheral membrane protein and is conjectured to function in a manner similar to plastocyanin. In the present work, the respiratory chain is further elucidated and the purification and characterization of the most abundant components cytochrome bc and cytochrome ba3 from the membrane fraction are described. The cytochrome bc complex consists of a 14 and an 18 kDa subunit in a 1:1 ratio, with heme c bound to the larger polypeptide. An Fe-S subunit similar to that found in eukaryotic bc complexes has not yet been identified. The second membrane complex carries two different heme groups of the ba3-type as well as copper. It contains two subunits of 36 and 40 kDa. This cytochrome ba3 binds carbon monoxide, a feature common to terminal oxidases. There is no spectroscopic evidence for a second terminal oxidase; hence, under the growth conditions chosen the respiratory chain of N. pharaonis appears to be unbranched. In addition to these cytochromes, a succinate dehydrogenase which is solubilized from the membrane by detergents was isolated. A cytochrome c which was isolated from the cytosol has an unusually high molecular weight and a redox potential of -142 mV. A second cytosolic protein, ferredoxin, was purified to homogeneity. A comparison of the redox potentials of the isolated proteins with those obtained from the native membrane allows the construction of a possible electron transfer chain.

Bacterial Outer Membrane Proteins↗