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D Oesterhelt

Publications and source records attributed to D Oesterhelt.

At least 55 records · Page 3Linked to original sources
PubMed1998

Dynamics of different functional parts of bacteriorhodopsin: H-2H labeling and neutron scattering.

We show that dynamics of specific amino acids within a protein can be characterized by neutron spectroscopy and hydrogen-deuterium labeling, and we present data on the motions of a selected set of groups within bacteriorhodopsin (BR), the retinal-based proton pump in the purple membrane of halophilic Archaea. Elastic incoherent neutron scattering experiments allow the definition of motions in the nano- to picosecond time scale and have revealed a dynamical transition from a harmonic to a softer, anharmonic atomic fluctuation regime in the global behavior of proteins. Biological activity in proteins is correlated with this transition, suggesting that flexibility is required for function. Elastic incoherent neutron scattering is dominated by H atom scattering, and to study the dynamics of a selected part of BR, fully deuterated purple membrane with BR containing H-retinal, H-tryptophan, and H-methionine was prepared biosynthetically in Halobacterium salinarum. These amino acids cluster in the functional center of the protein. In contrast to the protein globally, the thermal motions of the labeled atoms were found to be shielded from solvent melting effects at 260 K. Above this temperature, the labeled groups appear as more rigid than the rest of the protein, with a significantly smaller mean square amplitude of motion. These experimental results quantify the dynamical heterogeneity of BR (which meets the functional requirements of global flexibility), on the one hand, to allow large conformational changes in the molecule and of a more rigid region in the protein, on the other, to control stereo-specific selection of retinal conformations.

Amino Acids↗
PubMed1998

Structure and hydration of the M-state of the bacteriorhodopsin mutant D96N studied by neutron diffraction.

Neutron diffraction from oriented purple membrane fragments at various hydration levels, coupled with H2O/2H2O exchange, was used to compare the structure and hydration of the light-adapted initial state (B-state) and the M photointermediate of bacteriorhodopsin mutant D96N. Diffraction patterns were recorded at 86%, 75% and 57% relative humidity (r.h.). Structural changes observed at 86% and 75% r.h. are absent at 57% r.h., showing that they are uncoupled from the deprotonation of the Schiff base during formation of the M-state. In a current model, the M-state consists of two substates, M1 and M2. Our data suggest that the state trapped at 57% r.h. is M1 and that M2 is trapped at the higher r.h. values. The observed structural changes are, therefore, associated with the M1-->M2 transition, which can only take place at higher r.h. The difference Fourier projections of exchangeable hydrogen atoms and water molecules in the membrane plane are very similar for the B and M-states at 75% and 86% r.h. This shows that contrary to certain models, the structural changes in the M-state are not correlated with major hydration changes in the proton channel projection.

Asparagine↗
PubMed1998

Chloride- and pH-dependent proton transport by BR mutant D85N.

Photocurrents from purple membrane suspensions of D85N BR mutant adsorbed to planar lipid membranes (BLM) were recorded under yellow (lambda > 515 nm), blue (360 nm < lambda < 420 nm) and white (lambda > 360 nm) light. The pH dependence of the transient and stationary currents was studied in the range from 4.5 to 10.5. The outwardly directed stationary currents in yellow and blue light indicate the presence of a proton pumping activity, dependent on the pH of the sample, in the same direction as in the wild-type. The inwardly directed currents in white light, due to an inverse proton translocation, in a two-photon process, show a pH dependence as well. The stationary currents in blue and white light are drastically increased in the presence of azide, but not in yellow light. The concentration dependence of the currents on azide indicates binding of azide to the protein. In the presence of 1 M sodium chloride, the stationary proton currents in yellow light show an increase by a factor of 25 at pH 5.5. On addition of 50 mM azide, the stationary current in yellow light decreases again, possibly by competition between azide and chloride for a common binding site. The observed transport modes are discussed in the framework of the recently published IST model for ion translocation by retinal proteins [U. Haupts et al., Biochemistry 36 (1997) 2-7].

Azides↗
PubMed1998

Evidence for charge-controlled conformational changes in the photocycle of bacteriorhodopsin.

The existence of two different M-state structures in the photocycle of the bacteriorhodopsin mutant ASP38ARG was proved. At pH 6.7 (0 to -6 degreesC) a spectroscopic M intermediate (M1) that does not differ significantly in its tertiary structure from the light-adapted ground state accumulates under illumination. At pH > 9 another state (M2), characterized by additional pronounced changes in the Fourier transform infrared difference spectrum in the region of the amide I and II bands, accumulates. The M2 intermediate trapped at pH 9.6 displays the same changes in the x-ray diffraction intensities under continuous illumination as previously described for x-ray experiments with the mutant ASP96ASN. These observations indicate that in this mutant the altered charge distribution at neutral pH controls the tertiary structural changes that seem to be necessary for proton translocation.

Bacteriorhodopsins↗
PubMed1998

Low-temperature electron transfer from cytochrome to the special pair in Rhodopseudomonas viridis: role of the L162 residue.

Electron transfer from the tetraheme cytochrome c to the special pair of bacteriochlorophylls (P) has been studied by flash absorption spectroscopy in reaction centers isolated from seven strains of the photosynthetic purple bacterium Rhodopseudomonas viridis, where the residue L162, located between the proximal heme c-559 and P, is Y (wild type), F, W, G, M, T, or L. Measurements were performed between 294 K and 8 K, under redox conditions in which the two high-potential hemes of the cytochrome were chemically reduced. At room temperature, the kinetics of P+ reduction include two phases in all of the strains: a dominant very fast phase (VF), and a minor fast phase (F). The VF phase has the following t(1/2): 90 ns (M), 130 ns (W), 135 ns (F), 189 ns (Y; wild type), 200 ns (G), 390 ns (L), and 430 ns (T). These data show that electron transfer is fast whatever the nature of the amino acid at position L162. The amplitudes of both phases decrease suddenly around 200 K in Y, F, and W. The effect of temperature on the extent of fast phases is different in mutants G, M, L, and T, in which electron transfer from c-559 to P+ takes place at cryogenic temperatures in a substantial fraction of the reaction centers (T, 48%; G, 38%; L, 23%, at 40 K; and M, 28%, at 60 K), producing a stable charge separated state. In these nonaromatic mutants the rate of VF electron transfer from cytochrome to P+ is nearly temperature-independent between 294 K and 8 K, remaining very fast at very low temperatures (123 ns at 60 K for M; 251 ns at 40 K for L; 190 ns at 8 K for G, and 458 ns at 8 K for T). In all cases, a decrease in amplitudes of the fast phases is paralleled by an increase in very slow reduction of P+, presumably by back-reaction with Q(A)-. The significance of these results is discussed in relation to electron transfer theories and to freezing at low temperatures of cytochrome structural reorganization.

Amino Acid Sequence↗
PubMed1998

The structure and mechanism of the family of retinal proteins from halophilic archaea.

Retinal proteins from halophilic archaea provide a unique opportunity to analyze vectorial ion translocation. Studies on its structure, conformational changes, proton conduction and electrogenic steps have helped to elucidate the catalytic cycle of bacteriorhodopsin in increasing detail. Experimental modulation of the vectoriality and ion specificity by altering the substrate availability, point mutations and light conditions for the different retinal proteins allows the proposal of a general model of ion transport for this protein family.

Archaeal Proteins↗
PubMed1998

Localization of glycolipids in membranes by in vivo labeling and neutron diffraction.

Evidence is accumulating for the lateral organization of cell membrane lipids and proteins in the context of sorting or intracellular signaling. So far, however, information has been lacking on the details of protein-lipid interactions in such aggregates. Purple membranes are patches made up of lipids and the protein bacteriorhodopsin in the plasma membrane of certain Archaea. Naturally crystalline, they provide a unique opportunity to study the structure of a natural membrane at submolecular resolution by diffraction methods. We present a direct structural determination of the glycolipids with respect to bacteriorhodopsin in these membranes. Deuterium labels incorporated in vivo into the sugar moieties of the major glycolipid were localized by neutron diffraction. The data suggest a role for specific aromatic residue-carbohydrate stacking interactions in the formation of the purple membrane crystalline patches.

Amino Acids↗
PubMed1998

Regulation of switching frequency and bias of the bacterial flagellar motor by CheY and fumarate.

The effect of CheY and fumarate on switching frequency and rotational bias of the bacterial flagellar motor was analyzed by computer-aided tracking of tethered Escherichia coli. Plots of cells overexpressing CheY in a gutted background showed a bell-shaped correlation curve of Switching frequency and bias centering at about 50% clockwise rotation. Gutted cells (i.e., with cheA to cheZ deleted) with a low CheY level but a high cytoplasmic fumarate concentration displayed the same correlation of switching frequency and bias as cells overexpressing CheY at the wild-type fumarate level. Hence, a high fumarate level can phenotypically mimic CheY overexpression by simultaneously changing the switching frequency and the bias. A linear correlation of cytoplasmic fumarate concentration and clockwise rotation bias was found and predicts exclusively counter-clockwise rotation without switching when fumarate is absent. This suggests that (i) fumarate is essential for clockwise rotation in vivo and (ii) any metabolically induced fluctuation of its cytoplasmic concentration will result in a transient change in bias and switching probability. A high fumarate level resulted in a dose-response curve linking bias and cytoplasmic CheY concentration that was offset but with a slope similar to that for a low fumarate level. It is concluded that fumarate and CheY act additively presumably at different reaction steps in the conformational transition of the switch complex from counterclockwise to clockwise motor rotation.

Arabinose↗
PubMed1997

Chloride and proton transport in bacteriorhodopsin mutant D85T: different modes of ion translocation in a retinal protein.

Replacement of aspartate 85 (D85) in bacteriorhodopsin (BR) by threonine but not be asparagine creates at pH<7 an anion-binding site in the molecular similar to that in chloride pump halorhodopsin. Binding of various anions to BR-D85T causes a blue shift of the absorption maximum by maximally 57 nm. Connected to this color change is a change in the absorption difference spectrum of the initial state and the longest living photo intermediate from a positive difference maximum at 460 nm in the absence of transported anions to one at 630 nm in their presence. Increasing anion concentration cause decreasing decay times of this intermediate. At physiological pH, BR-D85T but not BR-D85N transports chloride ions inward in green light, protons outward in blue or green light and protons inward in white light (directions refer to the intact cell). The proton movements are observable also in BR-D85N. Thus, creation of an anion-binding site in BR is responsible for chloride transport and introduction of anion-dependent spectroscopic properties at physiological pH. The different transport modes are explained with the help of the recently proposed IST model, which states that after light-induced isomerization of the retinal an ion transfer step and an accessibility change of the active site follow. The latter two steps occur independently. In order to complete the cyclic event, the accessibility change, ion transfer and isomerization state have to be reversed. The relative rates of accessibility changes and ion transfer steps define ultimately the vectoriality of ion transfers. All transport modes described here for the same molecule can satisfactorily be described in the framework of this general concept.

Anions↗
PubMed1997

Specific arginine and threonine residues control anion binding and transport in the light-driven chloride pump halorhodopsin.

The light-driven chloride pump halorhodopsin (HR), a halobacterial retinal protein, was studied by comparing wild type with specific mutants. Changes of conserved arginine and threonine residues in the transmembrane regions could be classified in two categories: in the extracellular half of the molecule, mutations influence anion uptake and binding. R108 mutations abolish all anion effects previously attributed to two distinct binding sites and change the characteristic photochemistry. Neutral residues at position 108 completely inactivate the pump. T111 increases the affinity of this anion binding site without being essentially important. In the photochemical cycles of the mutants T111V and Q105E, a red-shifted absorbing intermediate is enriched indicating retarded anion uptake. On the cytoplasmic side, mutations do not change anion binding properties of the unphotolyzed protein, but slow down anion release thereby reducing the chloride transport activity and the photocycling rate. The lowest activity is found for T203V, while R200 mutations have weaker effects. Thus, in the symmetrically arranged pairs R108/T111 and T203/R200, threonine and arginine play different roles, reflecting high affinity anion uptake by the former and effective anion release catalyzed by the latter residues. A model for the anion transport mechanism in HR is suggested comprising the specific functions of channel-lining residues.

Anions↗
PubMed1997

Reconstitution of bacteriorhodopsin from the apoprotein and retinal studied by Fourier-transform infrared spectroscopy.

The reconstitution of the retinal-containing protein bacteriorhodopsin (BR) from the apoprotein and retinal has been studied by Fourier-transform infrared (FTIR) difference spectroscopy. 9-cis-Retinal which occupies the binding site but does not reconstitute the chromophore was used as "caged retinal". Photoisomerization to the all-trans isomer triggers the reconstitution reaction. Absorption bands in the FTIR difference spectra of the educt and product of the reaction could be assigned by comparison with a 9-cis-retinal FTIR spectrum or an FT-Raman spectrum of BR and due to band shifts observed upon deuterium exchange. Specific difference bands were assigned to the protonated carboxyl groups of D96 and D115 by use of the mutants D115N and D96N. Both aspartic acids are protonated also in the apoprotein with pKa values above 10 and undergo a frequency shift toward higher wavenumbers indicating a more hydrophobic environment in the reconstituted protein. No indication was found for protonation changes of carboxyl groups or other protonatable residues when carrying out the reaction at pH values between 4 and 10. The pH-dependent protonation changes reported earlier [Fischer & Oesterhelt (1980) Biophys. J. 31, 139-146] therefore may be caused by protons in a hydrogen-bonded network. Mutations of E204, but not of D38 or E9, cancel proton uptake during reconstitution at high pH as well as proton release at low pH. It is concluded, that E204, without changing its protonation state itself, is part of a protonatable hydrogen-bonded network which changes its pKa during reconstitution thereby causing the observed protonation changes.

Bacteriorhodopsins↗
PubMed1997

The tertiary structural changes in bacteriorhodopsin occur between M states: X-ray diffraction and Fourier transform infrared spectroscopy.

The tertiary structural changes occurring during the photocycle of bacteriorhodopsin (BR) are assigned by X-ray diffraction to distinct M states, M1 and M2. Purple membranes (PM) of the mutant Asp96Asn at 15, 57, 75 and 100% relative humidity (r.h.) were studied in a parallel X-ray diffraction and Fourier transform infrared (FTIR) spectroscopic investigation. Light-dependent conformational changes of BR-Asp96Asn are observed at high hydration levels (100 and 75% r.h.) but not in partially dehydrated samples (57 and 15% r.h.). The FTIR spectra of continuously illuminated samples at low and high hydration, despite some differences, are characteristic of the M intermediate. The changes in diffraction patterns of samples in the M2 state are of the same magnitude as those of wild-type samples trapped with GuaHCl in the M(G) state. Additional large changes in the amide bands of the FTIR spectra occur between M2 and M(G). This suggests, that the tertiary structural changes between M1 and M2 are responsible for the switch opening the cytoplasmic half-channel of BR for reprotonation to complete the catalytic cycle. These tertiary structural changes seem to be triggered by a charge redistribution which might be a common feature of retinal proteins also in signal transduction.

Amino Acid Sequence↗
PubMed1997

Electron diffraction studies of light-induced conformational changes in the Leu-93 --> Ala bacteriorhodopsin mutant.

We previously have presented evidence for prominent structural changes in helices F and G of bacteriorhodopsin during the photocycle. These changes were determined by carrying out electron diffraction analysis of illuminated two-dimensional crystals of wild-type bacteriorhodopsin or the Asp-96 --> Gly mutant that were trapped at a stage in the photocycle after light-driven proton release, but preceding proton uptake from the aqueous medium. Here, we report structural analysis of the long-lived O intermediate observed in the photocycle of the Leu-93 --> Ala mutant, which accumulates after the release and uptake of protons, but before the reisomerization of retinal to its initial all-trans state. Projection Fourier difference maps show that upon illumination of the Leu-93 --> Ala mutant, significant structural changes occur in the vicinity of helices C, B, and G, and to a lesser extent near helix F. Our results suggest that (i) all four helices that line the proton channel (B, C, F, and G) participate in structural changes during the late stages of the photocycle, and (ii) completion of the photocycle involves significant conformational changes in addition to those that are associated with steps in proton transport.

Bacteriorhodopsins↗
PubMed1997

The primary structure of the Cl(-)-translocating ATPase, b subunit of Acetabularia acetabulum, which belongs to the F-type ATPase family.

The genes possibly encoding the b subunit (50 kDa) of the Cl(-)-translocating ATPase of Acetabularia acetabulum were cloned from total RNA and from poly(A)+ RNA and sequenced. The deduced amino acid sequence of the open reading frame consisted of 478 amino acids and showed high similarity to the beta subunit of chloroplast F1-ATPase. Gene fragments encoding the putative beta subunit of chloroplast F1- (273 bp) and mitochondrial F1-ATPases (332 bp) were also cloned from A. acetabulum and sequenced, respectively. The deduced amino acid sequence of the chloroplast F1-ATPase showed 92.5% identity to be primary structure of the b subunit of the Cl(-)-translocating ATPase, while the nucleotide sequences were 79.9% identical. The deduced amino acid sequence of the latter was 77.3% identical to that of the b subunit of the Cl(-)-translocating ATPase and the nucleotide sequences were 67.5% identical. By Northern analysis, these three beta-like genes were demonstrated to be transcribed with different sizes of RNA species. A putative chloroplast F1-beta fragment also hybridized with chloroplast DNA isolated from the organism.

Acetabularia↗
PubMed1997

Very fast electron transfer from cytochrome to the bacterial photosynthetic reaction center at low temperature.

Electron transfer from the proximal heme c-559 to the primary donor P has been studied in reaction centers of the photosynthetic bacterium Rhodopseudomonas viridis in which the tyrosine residue L162 was replaced by threonine. In the wild type, when the two high-potential hemes of the tetraheme cytochrome are reduced before flash excitation, a rapid electron transfer (t1/2 = 190 ns) observed at ambient temperature disappears below 190 K. In the mutant, the reaction is partly maintained down to 8 K, leading to irreversible charge separation. The reaction rate is nearly temperature-independent between 294 K and 8 K (t1/2 approximately 450 ns). The different behavior of wild type and mutant reaction centers is attributed to differences in a network of water molecules, the freezing of which may block structural reorganizations associated with cytochrome oxidation, in the wild type but not in the mutant.

Cytochrome c Group↗
PubMed1997

General concept for ion translocation by halobacterial retinal proteins: the isomerization/switch/transfer (IST) model.

Bacteriorhodopsin (BR), which transports protons out of the cell in a light-driven process, is one of the best-studied energy-transducing proteins. However, a consensus on the exact molecular mechanism has not been reached. Matters are complicated by two experimental facts. First, recent results using BR mutants (BR-D85T) and the homologous protein sensory rhodopsin I demonstrate that the vectoriality of active proton transport may be reversed under appropriate conditions. Second, in BR-D85T as well as in the homologous halorhodopsin, protons and chloride ions compete for transport; e.g. the same molecule may transport either a positive or a negative ion. To rationalize these results, we propose a general model for ion translocation by bacterial rhodopsins which is mainly based on two assumptions. First, the isomerization state of the retinylidene moiety governs the accessibility of the Schiff base in the protein; e.g. all-trans, 15-anti, and 13-cis-15-anti direct the Schiff base to extracellular and cytoplasmic accessibility, respectively, but change in accessibility (called the "switch") is a time-dependent process in the millisecond time range. A light-induced change of the isomerization state induces not only a change in accessibility but also an ion transfer reaction. Second, we propose that these two processes are kinetically independent, e.g. that relative rate constants in a given molecule determine which process occurs first, ultimately defining the vectoriality of active transport.

Bacterial Proteins↗
PubMed1997

Expression of beta 2-adrenoceptors in halobacteria.

Halobacteria are halophilic representatives of the recently defined domain, the Archaea. Halobacterium salinarium belongs to this group of microorganisms and contains large amounts of bacteriorhodopsin in its membrane. Bacteriorhodopsin is a seven-transmembrane protein that consists of bacterio-opsin (BO), and the chromophore retinal, which is covalently attached to BO. We have investigated whether the expression machinery for BO can be utilized for synthesis of the human beta 2-adrenoceptor (beta 2-AR), a protein with a similar seven-transmembrane-helix topology. An expression vector for BO synthesis was modified to express beta 2-ARs under the control of BO regulatory clements in H. salinarium. Homologous recombination into the genome was verified by polymerase chain reactions. Northern blots revealed transcripts of the calculated size and significant amounts of epitope-tagged beta 2-ARs were detected in Western blots. However, binding of the beta-AR antagonist 125I-cyanopindolol revealed low levels of functional receptors, and the ligand binding properties of these receptors were altered when compared to native receptors. Expression of chimeras containing larger amino terminal portions of BO did not result in higher receptor levels. Expression of beta 2-AR in Haloferax volcanii, another member of halobacteria, was achieved with a vector carrying the ferredoxin promoter. The levels of functional receptor as determined by 125I-cyanopindolol binding were 180 fmol/mg protein. The beta-AR ligands isoprenaline and propranolol showed affinities expected for functional beta 2-ARs. Thus, functional human beta 2-ARs were expressed in halobacteria, constituting a first approach for expression of a eukaryotic protein in the domain of Archaea.

Adrenergic Antagonists↗