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PubMed2003

Crystal structure of halophilic dodecin: a novel, dodecameric flavin binding protein from Halobacterium salinarum.

A novel, 68 amino acid long flavoprotein called dodecin has been discovered in the proteome of Halobacterium salinarum by inverse structural genomics. The 1.7 A crystal structure of this protein shows a dodecameric, hollow sphere-like arrangement of the protein subunits. Unlike other known flavoproteins, which bind only monomeric flavin cofactors, the structure of the dodecin oligomer comprises six riboflavin dimers. The dimerization of these riboflavins along the re-faces is mediated by aromatic, antiparallel pi staggering of their isoalloxazine moieties. A unique aromatic tetrade is formed by further sandwiching of the riboflavin dimers between the indole groups of two symmetry-related Trp36s. So far, the dodecins represent the smallest known flavoproteins. Based on the structure and the wide spread occurrences in pathogenic and soil eubacteria, a function in flavin storage or protection against radical or oxygenic stress is suggested for the dodecins.

Amino Acid Sequence↗
PubMed2001

Structural stabilization of [2Fe-2S] ferredoxin from Halobacterium salinarum.

The ferredoxin of the extreme haloarchaeon Halobacterium salinarum requires high (>2 M) concentration of salt for its stability. We have used a variety of spectroscopic probes for identifying the structural elements which necessitate the presence of high salt for its stability. Titration of either the fluorescence intensity of the tryptophan residues or the circular dichroism (CD) at 217 nm with salt has identified a structural form at low (<0.1 M) concentration of salt. This structural form (L) exhibits increased solvent exposure of W side chain(s) and decreased level of secondary structure compared to the native (N) protein at high concentrations of salt. The L-form, however, contains significantly higher levels of both secondary and tertiary structures compared to the form (U) found in highly denaturing conditions such as 8 M urea. The structural integrity of the L-form was highly pH dependent while that of N- or U-form was not. The pH dependence of either fluorescence intensity or CD of the L-form showed the presence of two apparent pK values: approximately 5 and approximately 10. The structural integrity of the L-form at low (<5) pH was very similar to that of the N-form. However, titration with denaturants showed that the low pH L-form is significantly less stable than the N-form. The increased destabilization of the L-form with the increase in pH was interpreted to be due to mutual Coulombic repulsion of carboxylate side chains (pK approximately 6) and due to the disruption of salt bridge(s) between ionized carboxylates and protonated amino groups (pK approximately 10). Estimation of solvent accessibility of W residues by fluorescence quenching, and measurement of decay kinetics of fluorescence intensity and anisotropy strongly support the above model. Polylysine interacted stoichiometrically with the L-form of ferredoxin resulting in nativelike structure. In conclusion, our studies show that high concentration of salt stabilizes the haloarchaeal ferredoxin in two ways: (i) neutralization of Coulombic repulsion among carboxyl groups of the acidic residues, and (ii) salting out of hydrophobic residues leading to their burial and stronger interaction.

Circular Dichroism↗
PubMed1992

Chromophore of sensory rhodopsin II from Halobacterium halobium.

The photoreceptor sensory rhodopsin II (sR-II) was enriched 120-fold from cell membranes of Halobacterium halobium. The final preparation yields sR-II with a specific content of 3 nmol of sR-II/mg of protein. The spectroscopic measurements were performed on the enriched photoreceptor solubilized in digitonin. In the absolute absorption spectrum of the partially purified receptor, the main peak in the visible range corresponded to sR-II with a maximum at 488 nm. Cytochromes contributed to the spectrum only in a minor band at 415 nm. The extinction coefficient of sR-II was estimated from difference spectra during bleaching with hydroxylamine to be 48,000 M-1 cm-1. The reduced chromophore displayed a pronounced fine structure which is due to the coplanarity of the retinyl residue. The isomeric composition of the chromophore from the enriched photoreceptor was determined in retinal extracts in HPLC. The dark-adapted sR-II contains 80% all-trans- and 20% 13-cis-retinal. After illumination, the ratio changed to 1:1, indicating a trans-cis isomerization during the photocycle of sR-II.

Archaeal Proteins↗
PubMed1993

The effects of sulfite or nitrate on turnover-dependent inhibition in the ATPase from Halobacterium saccharovorum are related to the binding of the second metal ion.

The turnover-dependent inhibition of the Halobacterium saccharovorum ATPase is dependent on two parameters: pH and the concentration of the divalent cation present. At pH 6 and 1 mM Mn2+ the inhibition is small, but increases steeply with 6 mM Mn2+. In contrast, at pH 8.5 the inhibition is more than 90% at 1 mM Mn2+, and higher concentrations have little additional effect. A relationship between the occupation of a second metal ion binding site and turnover-dependent inhibition was postulated previously [Schobert, B. (1992) J. Biol. Chem. 267, 10252-10257]. The results lead to a model where this site (X-) can alternatively bind protons (XH), depending on the pH and the free metal ion concentration. The pKa of XH is estimated to be 9. The turnover-dependent inhibition is diminished by bisulfite, whereas sulfite is ineffective. The kinetics show that bisulfite and metal ion compete for the same site. In the proposed model, bisulfite binds via its negative charge to the site from which Pi was released and is arranged such as to interact with X- via its protonated group (X-HSO3-). In this way, formation of the inhibited enzyme species XMe is prevented. Inhibitory anions like nitrate, which carry a permanent dipole as a common feature, show uncompetitive inhibition vs metal ions. The data are compatible with a model in which these inhibitors bind to the vacant Pi site and position their positive charges near XH. As a consequence, the pKa of XH is decreased and X- is stabilized, which in turn favors the formation of XMe. The downshift in pKa was calculated to be 0.7 pH unit.

Adenosine Triphosphatases↗
PubMed1984

Nuclear magnetic resonance studies of amino acids and proteins. Deuterium nuclear magnetic resonance relaxation of deuteriomethyl-labeled amino acids in crystals and in Halobacterium halobium and Escherichia coli cell membranes.

We have obtained deuterium (2H) Fourier transform nuclear magnetic resonance (NMR) spectra of zwitterionic L-[beta-2H3]alanine, DL-[gamma-2H6]valine, DL-[beta, gamma-2H4]threonine, L-[delta-2H3]leucine, and L-[alpha, beta, gamma, gamma', delta-2H10]isoleucine in the crystalline solid state and have determined the deuteriomethyl group spin-lattice relaxation rates as a function of temperature. The results yield the Arrhenius activation energies (delta E) for methyl rotation, and through use of a suitable mathematical model, rotational correlation times, tau c. For alanine, valine, threonine, leucine, and isoleucine at 37 degrees C, tau c and delta E values are 780, 100, 40, 38, and 18 ps and 22, 14.0, 17.6, 15.5, and 8.6 kJ, respectively. For L-[beta-2H3]alanine in the zwitterionic lattice, a spin-lattice relaxation time (T1) minimum of 2.1 +/- 0.3 ms is observed (at 0 degree C), in excellent agreement with the 1.92-ms prediction of the mathematical model. Similar tau c and delta E measurements are reported for bacteriorhodopsin in the purple membrane of Halobacterium halobium R1 and for Escherichia coli cell membranes. Overall, our results demonstrate a great similarity between the dynamics in amino acid crystals and in membrane proteins. However, threonine exhibits a nonlinear Arrhenius behavior in bacteriorhodopsin, and in the valine-, leucine-, and isoleucine-labeled membrane samples at higher temperatures (approximately greater than 37 degrees C), there is evidence of an additional slow side-chain motion. The lipid phase state in E. coli does not appear to influence, on the average, the dynamics of the valine side chains. These results indicate that the sensitivity of the deuterium NMR technique is now adequate to study in moderate detail the dynamics of most types of amino acids in a membrane protein and that adequate sensitivity, in some instances, should be available for the study of individual amino acids in suitably labeled membrane proteins.

Amino Acids↗
PubMed1984

Interaction of aromatic retinal analogues with apopurple membranes of Halobacterium halobium.

Absorption spectral properties of aromatic analogues of retinal with apopurple membrane of Halobacterium halobium were studied. The spectra of the all-trans forms were composed of two or more absorption bands. During incubation at 20 degrees C, an absorption band above 500 nm increased in intensity gradually at the expense of an absorption band in the shorter wavelength region with no isomerization of the chromophore. The longer wavelength species was shown to be the protonated form of the shorter wavelength species by changing the pH of the medium. Upon irradiation with blue light, the bandwidth of the spectrum became smaller with isomerization of the chromophore to its 13-cis form. Irreversible binding of protons on the membrane occurred during this process. The rate of the increase in the longer wavelength absorption band was especially low in the reaction with the all-trans form of retinal analogues having a bulky substituent at the para or meta positions of the phenyl ring. In contrast, the 13-cis isomer of aromatic retinal analogues gave a single absorption peak. The extent of the spectral shift upon binding to apopurple membranes was compared over a series of aromatic retinals, and the results were explained in terms of steric interactions of the chromophore with the protein.

Bacteriorhodopsins↗
PubMed1980

Proton and carbon-13 nuclear magnetic resonance studies of the polar lipids of Halobacterium halobium.

The thermotropic behavior and the dynamic properties of the polar lipids of Halobacterium halobium were studied by 1H and 13C NMR. The studies were performed on three different preparations: micellar solutions in deuterated chloroform, multilamellar dispersions in 2H2O, and unilamellar vesicles obtained from the latter by ultrasonication. Due to the methyl side groups in the alkyl chains of these lipids, the 13C spectra are highly resolved in particular in the micellar solution, allowing peak assignment of the alkyl chain carbons. Proton and 13C T1 relaxation and line width measurements were made for all three preparations. In both lamellar dispersions, sharp discontinuities in these parameters were observed around 35 degrees C. This behavior is attributed to an abrupt change in the dynamics of the lipids within the membrane. The variation of the specific relaxation rate 1/NT1 of the 13C nuclei along the hydrocarbon chains of the H. halobium lipids exhibits maxima at the tertiary carbons to which the methyl side groups are attached. These maxima are particularly pronounced in the lamellar phases and suggest that the segmental motion ("kink" formation) at the tertiary carbons is hindered by the presence of the methyl groups.

Halobacterium↗
PubMed1980

Action spectrum and quantum efficiency for proton pumping in Halobacterium halobium.

The action spectrum and quantum efficiency (phi H+) for proton ejection from Halobacterium halobium have been determined under conditions chosen to minimize light-triggered proton influx which is usually observed in intact cells. The action spectrum for the carotenoid-containing strain, R1, shows that light energy absorbed by the carotenoids does not contribute to the proton ejection. After correction for shielding by the carotenoids and other cell pigments, the action spectrum closely follows the absorption spectrum of bacteriorhosopsin. Values determined for phi H+ in H. halobium cells and cell envelopes range from 0.4 to 0.7. These values are significantly higher than the currently accepted value for the quantum efficiency for the photoreaction cycle of bacteriorhodopsin in isolated purple membrane, suggesting that at least in intact cells and envelopes more than one proton is pumped during the bacteriorhodopsin photocycle. A new nondestructive assay for bacteriorhopopsin in intact cells and envelopes which also contain other pigments was used in this work.

Bacteriorhodopsins↗
PubMed1978

Phase transitions of the purple membranes of Halobacterium halobium.

Purple membranes of Halobacterium halobium were studied by differential scanning calorimetry. No transition was detected at temperatures below 70 degrees C. A small endothermic transition was seen at about 80 degrees C and a larger one at 100 degrees C. The larger transition is the irreversible denaturation of bacteriorhodopsin. The smaller transition is accompanied by a change in the visible absorption spectrum and is believed to be reversible, involving a cooperative change in crystalline structure of the membrane.

Bacteriorhodopsins↗
PubMed1978

Phase behavior of lipids from Halobacterium halobium.

Mixtures of dipalmitoylphosphatidylcholine with purple membrane lipids, red membrane lipids, or total lipids of Halobacterium halobium have been studied with differential scanning calorimetry. A comparison of red and purple membrane lipids reveals no difference in their phase behavior, indicating that lipid phase behavior plays no role in the in vivo separation of red and purple membranes. The effects of variation of the salt content of the suspending solution have also been examined. Studies of the melting behavior of these mixtures as H. halobium lipid content is varied suggest that the gel to liquid-crystal transition does not occur in the lipids of H. halobium.

Bacteriorhodopsins↗
PubMed1977

Light-activated amino acid transport systems in Halobacterium halobium envelope vesicles: role of chemical and electrical gradients.

The accumulation of 20 commonly occurring L-amino acids by cell envelope vesicles of Halobacterium halobium, in response to light-induced membrane potential and an artificially created sodium gradient, has been studied. Nineteen of these amino acids are actively accumulated under either or both of these conditions. Glutamate is unique in that its uptake is driven only by a chemical gradient for sodium. Amino acid concentrations at half-maximal uptake rates (Km) and maximal transport rates (Vmax) have been determined for the uptake of all 19 amino acids. The transport systems have been partially characterized with respect to groups of amino acids transported by common carriers, cation effects, and relative response to the electrical and chemical components of the sodium gradient, the driving forces for uptake. The data presented clearly show that the carrier systems, which are responsible for uptake of individual amino acids, are as variable in their properties as those found in other organisms; i.e., some are highly specific for individual amino acids, some transport several amino acids competitively, some are activated by a chemical gradient of sodium only, and some function also in the complete absence of such a gradient. For all amino acids, Na+ and K+ are both required for maximal rate of uptake. The carriers for L-leucine and L-histidine are symmetrical in that these amino acids are transported in both directions across the vesicle membrane. It is suggested that coupling of substrate transport to metabolic energy via transient ionic gradients may be a general phenomenon in procaryotes.

Amino Acids↗
PubMed1976

Photoreceptor protein from the purple membrane of Halobacterium halobium. Molecular weight and retinal binding site.

The apparent molecular weight of the purple membrane protein of Halobacterium halobium was found to be 20 000 by sodium dodecyl sulfate gel electrophoresis and by gel filtration in sodium dodecyl sulfate. However, the molecular weight value determined by gel filtration in 6 M guanidine was 28 000. To resolve this discrepancy, methods insensitive to or independent of the conformation of the protein were used to estimate the molecular weight. Analytical ultracentrifugation of the sodium dodecyl sulfate-protein complex, peptide mapping, and amino acid analysis all gave values of 25 000 +/- 1000, a figure in agreement with a recent x-ray study. Borohydride reduction was used to attach the retinal cofactor covalently to a lysine residue. After digestion with thermolysin, peptide maps were prepared of the protein labeled at lysine residues with [14C] succinic anhydride both before and after reduction. Comparison of the maps showed one radioactive peptide with changed mobility. This peptide was isolated and shown to have the sequence Val-Ser-Asp-Pro-Asp-Lys-Lys with only one of the two lysine residues alkylated. Solid-phase sequencing showed the succinyl group to be at position 6 and hence the retinal group to be at position 7. It was possible that a small amount of retinal was also bound to Lys-6. There was no apparent homology with the corresponding peptide of vertebrate rhodopsin. No evidence of chain heterogeneity was found by radiochemical peptide mapping and sequence analysis of peptides containing lysine residues indicating that all protein chains of purple membrane are very similar or identical.

Amino Acid Sequence↗
PubMed1976

Light-induced glutamate transport in Halobacterium halobium envelope vesicles. I. Kinetics of the light-dependent and the sodium-gradient-dependent uptake.

During illumination Halobacterium halobium cell envelope vesicles accumulate [3H]glutamate by an apparently unidirectional transport system. The driving force for the active transport originates from the light-dependent translocation of protons by bacteriorhodopsin and is due to a transmembrane electrical potential rather than a pH difference. Transport of glutamate against high concentration gradients is also achieved in the dark, with high outside/inside Na+ gradients. Transport in both cases proceeds with similar kinetics and shows a requirement for Na+ on the outside and for K+ on the inside of the vesicles. The unidirectional nature of glutamate transport seems to be due to the low permeability of the membranes to the anionic glutamate, and to the differential cation requirement of the carrier on the two sides of the membrane for substrate translocation. Thus, glutamate gradients can be collapsed in the dark either by lowering the intravesicle pH (with nigericin, or carbonyl cyanide p-trifluoromethoxyphenylhydrazone plus valinomycin), or by reversing the cation balance across the membranes, i.e., providing NaCl on the inside and KCl on the outside of the vesicles. In contrast to the case of light-dependent glutamate transport, the initial rates of Na+-gradient-dependent transport are not depressed when an opposing diffusion potential is introduced by adding the membrane-permeant cation, triphenylmethylphosphonium bromide. Therefore, it appears that, although the electrical potential must be the primary source of energy for the light-dependent transport, the translocation step itself is electrically neutral.

Biological Transport, Active↗
PubMed1976

Light-induced membrane potential and pH gradient in Halobacterium halobium envelope vesicles.

Illumination of envelope vesicles prepared from Halobacterium halobium cells causes translocation of protons from inside to outside, due to the light-induced cycling of bacteriorhodopsin. This process results in a pH gradient across the membranes, an electrical potential, and the movements of K+ and Na+. The electrical potential was estimated by following the fluorescence of a cyanine dye, 3,3'-dipentyloxadicarbocyanine. Illumination of H. halobium vesicles resulted in a rapid, reversible decrease of the dye fluorescence, by as much as 35%. This effect was not seen in nonvesicular patches of purple membrane. Observation of maximal fluorescence decreases upon ilumination of vesicles required an optimal dye/membrane protein ratio. The pH optimum for the lightinduced fluorescence decrease was 6.0. The decrease was linear with actinic light intensity up to about 4 X 10(5) ergs cn-2 s-1. Valinomycin, gramicidin, and triphenylmethylphosphonium ion all abolished the fluorescence changes. However, the light-induced pH change was enhanced by these agents. Conversely, buffered vesicles showed no pH change but gave the same or larger fluorescence changes. Thus, we have identified the fluorescence decrease with a light-induced membrane potential, inside negative. By using valinomycin-K+-induced membrane potentials, we calibrated the fluorescence decrease with calculated Nernst diffusion potentials. We found a linear dependence between potential and fluorescence decrease of 3 mV/%, up to 90 mV. When the envelope vesicles were illuminated, the total proton-motive force generated was dependent on the presence of Na+ and K+ and their concentration gradients across the membrane. In general, K+ appeared to be more permeable than Na+ and, thus, permitted development of greater pH gradients and lower electrical potentials. By calculating the total proton-motive force from the sum of the pH and potential terms, we found that the vesicles can produce proton-motive forces near--200 mV.

Cell Membrane↗
PubMed1976

Existence of electrogenic hydrogen ion/sodium ion antiport in Halobacterium halobium cell envelope vesicles.

Illumination causes the extrusion of protons from Halobacterium halobium cell envelope vesicles, as a result of the action of light on bacteriorhodopsin. The protonmotive force developed is coupled to the active transport of Na+ out of the vesicles. The light-dependent ion fluxes in these vesicles were studied by following changes in the external pH, in the fluorescence of the dye, 3,3'-dipentyloxadicarbocyanine, in the 22Na content of the vesicles, and in [3H]dibenzyldimethylammonium (DDA+) accumulation. During Na+ efflux, and dependent on the presence of Na+ inside the vesicles, the initial light-induced H+ extrusion is followed by H+ influx, which results in net alkalinization of the medium at pH greater than 6.5. When the Na+ content of the vesicles is depleted, the original net of the medium is restored and large deltapH develops, accompanied by a decrease in the electrical potential. Data reported elsewhere suggest that the driving force for the transport of some amino acids consists mainly of the electrical potential, while for others it comprises the Na+ gradient as well. Glutamate transport appears to be energized only by the Na+ gradient. The development of the Na+ gradient during illumination thus plays an important role in energy coupling. The results obtained are consistent with the existence of an electrogenic H+/Na+ antiport mechanism (H+/Na+ greater than 1) in H halobium which facilitates the uphill Na+ efflux. The light-induced protonmotive force thereby becomes the driving force in forming a Na+ gradient. The presence of the proposed H+/Na+ antiporter explains many of the light-induced pH effects in intact H. halobium cells.

Biological Transport, Active↗
PubMed2002

Demonstration of 2:2 stoichiometry in the functional SRI-HtrI signaling complex in Halobacterium membranes by gene fusion analysis.

A fusion protein in which the C-terminus of Halobacterium salinarum sensory rhodopsin I (SRI) is connected by a flexible linker to the N-terminus of its transducer (HtrI) was constructed and expressed in H. salinarum. The fusion protein mediated attractant responses to orange light and repellent responses to UV/violet light that were comparable to those produced by the wild-type SRI-HtrI complex. Immunoblot analysis of H. salinarum membrane proteins demonstrated intact fusion protein and no detectable proteolytic cleavage products. Rapid oxidative cross-linking of a monocysteine mutant in the HtrI domain confirmed that the fusion protein exists as a homodimer in the membrane. HtrI-free SRI and HtrI-complexed SRI have been shown previously to exhibit large differences in the pH dependence of their photocycle kinetics and in the pK(a) of Asp76 that controls a pH-dependent spectral transition in SRI. These differences were used to assess whether only one or both SRI domains in the fusion protein were complexed properly to the HtrI homodimer. Measurement of the photochemical activity, the photocycle kinetics, and the absorption spectra at various pH values established that both SRI domains are complexed to HtrI in the fusion protein, and therefore the stoichiometry is 2:2. Closer examination of the HtrI effect on SRI revealed that Asp76 titration in HtrI-free SRI fits two pK(a) values, with 98% and 2% of the molecules titrating with pK(a)'s of 7 and 9, respectively. The same two pK(a)'s of Asp76 are evident in HtrI-complexed SRI, but with 13% with pK(a) of 7 and 87% with pK(a) of 9 and a similar bias toward the pK(a) of 9 in the fusion protein. Titration of the fusion protein with Ala substitution at Arg73, a residue in the photoactive site, in the SRI domain indicates that a basic residue at Arg73 is necessary for the lower pK(a) to be observed. A model in which Arg73 plays a role in the HtrI effect on SRI is discussed.

Archaeal Proteins↗
PubMed1996

Lipid-induced conformational changes of an integral membrane protein: an infrared spectroscopic study of the effects of Triton X-100 treatment on the purple membrane of Halobacterium halobium ET1001.

Exposure of purple membrane from Halobacterium halobium to sublytic concentrations of Triton X-100 results in significant changes in the bacteriorhodopsin (BR) photocycle (Mukhopadhyay et al., 1994). Infrared spectra of purple membrane samples exposed briefly to Triton indicate that this change in protein function accompanies the preferential release of purple membrane glycolipids and squalenes, an association of Triton with purple membrane, and a perturbation of specific lipid headgroup interactions within the membrane. Specifically, the bilayer alterations induced by Triton entail a disruption of lipid headgroup hydrogen bonding in addition to protein conformational changes involving a loss in beta-turn and alphaII-helical structures in BR. We propose that the purple membrane glycolipids and squalenes are critical for the normal functioning of the BR photocycle and that perturbations of these lipids cause the profound photocycle changes induced by exposure to Triton. Lipid reconstitution studies demonstrated that although several of the infrared spectral parameters characteristic of the structural changes induced by Triton were reversed, the photocycle characteristics of BR in native purple membrane were not regained. The observed changes in the vibrational spectra induced by lipid-mediated bilayer perturbations suggest a useful approach for clarifying structure-function relationships of intrinsic membrane proteins exhibiting transmembrane helices.

Bacteriorhodopsins↗
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↗