Biomedical subjects

D Oesterhelt

Publications and source records attributed to D Oesterhelt.

At least 91 records · Page 5Linked to original sources

PubMed1995

Mechanism of photosensory adaptation in Halobacterium salinarium.

Phototaxis in Halobacterium salinarium is the result of an interplay of sensory rhodopsin excitation and adaptation to the stimulus background. Adaptation to orange light, received by sensory rhodopsin I was probed by measuring the behavioral response of cells to a step-like decrease in intensity. Cells were able to adapt to an intensity range of more than four orders of magnitude. The data were analysed on the basis of theoretical fluence rate response relationships calculated from the photocycle kinetics of the complex of sensory rhodopsin I with its transducer HtrI. Independent of the stimulus background, the cellular response was shown to be a function of the absolute number of photoreceptor complex molecules turned over by the light stimulus. Receptor deactivation was identified as the underlying mechanism of adaptation and was sufficient to account for the experimental results. We suggest that reversible methylation of the transducer protein HtrI provides the chemical mechanism of sensory adaptation in H. salinarium and also explains the different sensitivity of the cells to orange and UV light.

Adaptation, Physiological↗

PubMed1995

Chemotaxis and phototaxis require a CheA histidine kinase in the archaeon Halobacterium salinarium.

Histidine kinases are part of the two-component signal transduction system responsible for eubacterial responses to diverse environmental signals. They have recently been detected in eukaryotes but their existence in the kingdom Archaea remains uncertain. Here we report the sequence and function of a histidine kinase (CheAH.s.) from Halobacterium salinarium, the first such transmitter in Archaea. The protein CheAH.s. (668 residues) has significant sequence identity with the CheA proteins known from eubacterial signal transduction (e.g. 34% identity with CheA from Bacillus subtilis). Antibodies were raised against CheAH.s. as expressed in Escherichia coli and were used in Western blotting to demonstrate the expression of cheAH.s. in H. salinarium. As has been observed for other halophilic proteins, CheAH.s. has a deviant electrophoretic migration, with an apparent molecular weight of 103 kDa on SDS-PAGE compared with a calculated molecular weight of 72 kDa. Deletion of a part of the cheAH.s. gene leads to loss of both chemotactic and phototactic responses in H. salinarium as measured by swarm plate assays, motion analysis and tethering experiments. This indicates that CheAH.s. plays a crucial role in chemical and light signal integration, presumably interacting with at least two phototransducers and a number of chemoreceptors.

Amino Acid Sequence↗

PubMed1994

Sensory rhodopsin I photocycle intermediate SRI380 contains 13-cis retinal bound via an unprotonated Schiff base.

Sensory rhodopsin I (SRI), the mutated derivative SRI-D76N and the complex of SRI with its transducer HtrI were overexpressed in Halobacterium salinarium and analyzed by resonance Raman spectroscopy. In the initial state SRI contains all-trans retinal bound via a protonated Schiff base as confirmed by retinal extraction which yields 95 +/- 3% all-trans retinal. The photocycle intermediate absorbing maximally at 380 nm (SRI380) contains a Schiff base linkage between the protein and 13-cis retinal. Extraction of illuminated SRI yields up to 93% 13-cis retinal. Neither the mutation D76N nor HtrI changed the vibrational pattern of the chromophore.

Bacteriorhodopsins↗

PubMed1994

A cytoplasmic domain is required for the functional interaction of SRI and HtrI in archaeal signal transduction.

Phototaxis in the archaeon Halobacterium salinarium is mediated by a stable complex of the photoreceptor sensory rhodopsin I and its transducer HtrI, which relays the light stimulus to the signalling pathway. Removal of the cytoplasmic signalling domain of HtrI eliminated the SRI-specific motor response to light stimulation and led to the loss of the spectroscopically detectable physical interaction of SRI and HtrI. A similar phenotype was obtained by deleting part of a cytoplasmic loop located between the second transmembrane helix of HtrI and the signalling domain. These results indicate that the photochemical behavior of sensory rhodopsin I is not determined by interaction with the transmembrane helices of HtrI per se but functionally coupled to the signalling domain. It is proposed that light excitation of SRI results in a conformational change of the transducer which is conducted by the cytoplasmic loop, an extra module not found in the eubacterial transducer homologues, and activates the signalling domain.

Archaeal Proteins↗

PubMed1994

Purification and characterization of oxygen-evolving photosystem II core complexes from the green alga Chlamydomonas reinhardtii.

Oxygen-evolving photosystem II complexes were isolated from the green alga Chlamydomonas reinhardtii by selective solubilization of thylakoid membranes with dodecyl maltoside followed by density gradient centrifugation and anion-exchange chromatography. In the presence of CaCl2 and K3[Fe(CN)6] the complexes evolved oxygen at rates exceeding 1000 mumol (mg of chl)-1 h-1. The particles contained 40 chlorophylls a and had properties very similar to those of PSII isolated from higher plants. Chlamydomonas reinhardtii is now the first organism which can be used for both site-directed mutagenesis and detailed biochemical and biophysical characterization of oxygen-evolving photosystem II. It seems therefore to be an ideal model organism for investigation of structure-function relationships in photosynthetic oxygen evolution.

Animals↗

PubMed1994

Photosynthetic deficiency of a pufX deletion mutant of Rhodobacter sphaeroides is suppressed by point mutations in the light-harvesting complex genes pufB or pufA.

The pufX gene of the facultative phototroph Rhodobacter sphaeroides encodes a membrane protein that is required for photoheterotrophic growth. Deletion of pufX impairs the photosynthetic generation of a transmembrane potential, suggesting a role for the PufX protein in light-driven cyclic electron transfer [Farchaus, J. W., et al. (1992) EMBO J. 11, 2779-2788]. Here we describe the isolation and characterization of 65 spontaneous suppressor mutants in which photosynthetic competence was restored by secondary mutations. Genetic analysis revealed the occurrence of single point mutations altering highly conserved residues within the light-harvesting complex, B875. One of three tryptophan codons was changed to stop or arginine codons in 89% of these suppressor mutants. Spectral characterization and Western blot analysis were used to examine the B875 assembly and the stable expression of the altered light-harvesting polypeptides. Three different groups of suppressor mutants were found: (1) No stable expression of altered B875 polypeptides was detected for the alpha 43W-->* and beta 44W-->* mutants. (2) There was expression of the mutated B875-beta chain, but no stable B875 assembly in the beta 47W-->R mutants. (3) Intact B875 complexes were found for the alpha 47S-->F or beta 20H-->R mutants. These results provide evidence that the differently altered B875 polypeptides do not substitute directly for the PufX protein but lead to structural rearrangements in the macromolecular membrane organization, thus restoring a sufficiently high capacity for light-driven cyclic electron transfer.

Amino Acid Sequence↗

PubMed1994

Proton migration along the membrane surface and retarded surface to bulk transfer.

Since the proposal of the chemiosmotic theory there has been a continuing debate about how protons that have been pumped across membranes reach another membrane protein that utilizes the established pH gradient. Evidence has been gathered in favour of a 'delocalized' theory, in which the pumped protons equilibrate with the aqueous bulk phase before being consumed, and a 'localized' one, in which protons move exclusively along the membrane surface. We report here that after proton release by an integral membrane protein, long-range proton transfer along the membrane surface is faster than proton exchange with the bulk water phase. The rate of lateral proton diffusion can be calculated by considering the buffer capacity of the membrane surface. Our results suggest that protons can efficiently diffuse along the membrane surface between a source and a sink (for example H(+)-ATP synthase) without dissipation losses into the aqueous bulk.

Arylsulfonates↗

PubMed1994

Influence of M subunit Thr222 and Trp252 on quinone binding and electron transfer in Rhodobacter sphaeroides reaction centres.

M subunit Trp252 is the only amino acid residue which is located between the bacteriopheophytin HA and the quinone QA in the photosynthetic reaction centre of Rhodobacter sphaeroides. Oligodeoxynucleotide-directed mutagenesis was employed to elucidate the influence of this aromatic amino acid on the electron transfer between these two chromophores. For this, M subunit Trp252 was changed to tyrosine or phenylalanine, and Thr222, which presumably forms a hydrogen bridge to the indole ring of M subunit Trp252, to valine. In all three mutated reaction centres, the electron-accepting ubiquinone QA is less firmly bound to its binding site than in the wild-type protein. The electron transfer from the reduced bacteriopheophytin HA- to QA proceeds in the wild-type and in the mutant ThrM222Val within 220 ps. However, in the mutants TrpM252Tyr and TrpM252Phe the time constants are 600 ps and 900 ps, respectively. This indicates that M subunit Trp252 participates in the binding of QA and reduction of this quinone.

Base Sequence↗

PubMed1994

Identification of volatile forms of methyl groups released by Halobacterium salinarium.

halobacterium salinarium (formerly H. halobium) is a chemotactic and phototactic archaeon from which volatile methyl groups are released continually, a phenomenon related to its sensory system. We found that released methyl groups comprised two different chemical species, methanol and methanethiol, the sulfur analog of methanol. Radiolabeling experiments showed that the methyl groups of both compounds, as well as the sulfur of methanethiol, were derived from methionine but were donated to cellular components and subsequently cleaved to produce the respective volatile compounds. Previous work had shown that chemostimuli and photostimuli result in transient increases in the rate of release of volatile methyl groups. We found that these increases reflected increased release of methanol but not of methanethiol. Thus, the methyl group chemistry of the H. salinarium sensory system is analogous to the well-studied chemotactic system of Escherichia coli. The reactions that result in methanethiol release are of unknown function and have unusual features. They may involve a methionine-gamma-lyase activity we detected in H. salinarium. Sulfur derived from methionine was found attached to specific proteins in reduction-sensitive disulfide linkages.

Carbon-Sulfur Lyases↗

PubMed1994

The primary structure of halocyanin, an archaeal blue copper protein, predicts a lipid anchor for membrane fixation.

Halocyanin, a small blue copper protein, was isolated from the haloalkaliphilic archaeon Natronobacterium pharaonis. The NH2 terminus was not accessible to Edman degradation. About 70% of the amino acid sequence was determined by protein sequence analysis. The sequence information of two peptides was used for cloning and sequencing the halocyanin gene (hcy). The open reading frame codes for 489 base pairs, which account for a protein with 163 amino acids and a molecular mass of 17,223 Da. The discrepancy between this value and the molecular mass of 15,456 +/- 1.5 Da for the copper-free protein determined by electrospray mass spectrometry can be explained by a post-translational processing of the gene product. The NH2-terminal sequence of the open reading frame contains a motif that is characteristic for prokaryotic lipoproteins. Assuming a similar processing for halocyanin, Cys at position 25 of the primary transcript would be modified by a diphytanyl (glycerol)diether. Subsequently, the precursor is cleaved by a signal peptidase II-like protease and then acetylated at its NH2-terminal alpha-amino group. These modifications would yield a protein with a calculated molecular mass of 15,456 Da. A comparison of the primary structure of halocyanin with a number of other blue copper proteins places it into the plastocyanin-related group.

Amino Acid Sequence↗

PubMed1994

Lowering the intrinsic pKa of the chromophore's Schiff base can restore its light-induced deprotonation in the inactive Tyr-57-->Asn mutant of bacteriorhodopsin.

Following light absorption, at neutral pH the bacteriorhodopsin mutant Y57N does not show Schiff base deprotonation (no M intermediate) or proton pumping activity. We reasoned that this might be due to improper delta pKa between the proton-donating Schiff base and the proton-accepting Asp-85 after light absorption. To test this, we reduced the intrinsic pKa of the protonated Schiff base in the pigment (and thus in the photointermediates) by replacing the retinal chromophore with an analogue, 14-F retinal. This substitution restores light-induced M formation, strongly suggesting that light-induced Schiff base deprotonation is accomplished by lowering its pKa during the photochemical cycle. Thus, while it is generally accepted that the Schiff base deprotonation during the photocycle takes place because of the light-induced reduction in its pKa, we provide here the first experimental evidence of this phenomenon.

Amino Acid Sequence↗

PubMed1994

Phototaxis of Halobacterium salinarium requires a signalling complex of sensory rhodopsin I and its methyl-accepting transducer HtrI.

Sensory rhodopsin I (SRI) is a photoreceptor that mediates phototaxis in the archaeon Halobacterium salinarium. Receptor excitation is relayed to the motility system of the cell by the methyl-accepting transducer protein HtrI. In membranes prepared from cells that lack HtrI the absorbance difference maximum of SRI was shifted from 587 to 565 nm. The thermal decay of the metastable photocycle intermediate SRI373 was measured as time-dependent recovery of the absorbance at 590 nm. In the absence of HtrI the decay was slowed down by two orders of magnitude. When SRI was overproduced in cells that contained normal levels of HtrI, the decay of SRI373 was biexponential indicating two kinetically distinct species. Spectroscopic measurements on intact cells revealed the same effect of HtrI on SRI photocycling as found in isolated membranes. By transient exposure of membranes from wild-type cells to low ionic strength, the decay of SR373 was slowed to the same value found for untreated membranes in the absence of HtrI. In parallel, the absorbance difference maximum was shifted to 565 nm indicating that a physical interaction of HtrI and SRI had been irreversibly destroyed. Overproduction of SRI in the presence of wild-type amounts of HtrI did not increase the light sensitivity of the cells to orange light step down stimulation. It is concluded that SRI and HtrI form a stable complex in the cell membrane that signals to the flagellar motor and defines absorbance maximum, photocycling rate and photochemical efficiency of SRI.

Archaeal Proteins↗

PubMed1994

Flash-induced electrogenic reactions in the SA(L223) reaction center mutant in Rhodobacter sphaeroides chromatophores.

The charge transfer events in the SA(L223) reaction center mutant Rhodobacter sphaeroides chromatophores were investigated by direct electrometry. Besides the primary charge separation, the small stigmatellin-sensitive electrogenic reaction due to the electron transfer from the primary to the secondary quinone acceptor in the reaction center complex was observed after the first flash. The second flash-induced electrogenic phase of the secondary quinone protonation and subsequent electrogenic reactions of the cytochrome bc1 complex were much slower than those in chromatophores of the wild type. It is suggested that replacement of Ser-L223 by Ala impairs both specific proton-conducting pathways leading to the secondary quinone QB.

Alanine↗

PubMed1994

Bacteriorhodopsin can function without a covalent linkage between retinal and protein.

Light energy is transferred from retinal to the protein in bacteriorhodopsin after absorption of a photon resulting in changes of protein conformation. To examine whether the covalent bond, formed by the carbonyl group of retinal and the epsilon-amino group of lysine 216, is essential for this process, a mutant with lysine 216 replaced by alanine was expressed in Halobacterium salinarium L33 (BO-, retinal+). Reconstitution of the chromoprotein with varying retinylidene-n-alkylamines was possible in isolated membranes as well as in whole cells. When the protein in membranes with retinylidene Schiff bases of n-alkylamines of different lengths was reconstituted, the most stable chromoprotein was formed with retinylideneethylamine. The absorbance maximum was at 475 nm in alkaline solution and 620 nm in acidic solution. At neutral pH values both species equilibrate with a third one absorbing maximally at 568 nm. Reconstitution of whole cells with retinylideneethylamine led to a specific proton pump activity of 30 mol of protons per mol of BR per minute. This value indicates a lower limit of transport; no light saturation could be reached in these measurements in contrast to wild-type BR where transport activities of 162 mol of protons per mol of BR per minute under identical conditions can be achieved. Action spectra from flash photolysis experiments revealed that only the 568-nm form led to a M-intermediate with a half-time of decay of 17 ms. In summary, it could be shown that the covalent linkage between retinal and the protein is basically not required for the function of bacteriorhodopsin as a light-driven proton pump.

Bacteriorhodopsins↗

PubMed1994

Inversion of proton translocation in bacteriorhodopsin mutants D85N, D85T, and D85,96N.

Proton translocation activity of bacteriorhodopsin mutants lacking the proton acceptor Asp-85 was investigated using the black lipid membrane technique. Mutants D85N, D85T, and D85,96N were constructed and homologously expressed in Halobacterium salinarium to yield a membrane fraction with a buoyant density of 1.18 g/cm3, i.e., identical to that of wild-type purple membrane. In all mutants, the absorbance maximum was red-shifted between 27 and 49 nm compared with wild type, and the pKa values of the respective Schiff bases were reduced to between 8.3 and 8.9 compared with the value of > 13 in wild type. Therefore, a mixture of chromophores absorbing at 410 nm (deprotonated form) and around 600 nm (protonated form) exists at physiological pH. In continuous blue light, the deprotonated form generates stationary photocurrents. The currents are enhanced by a factor of up to 50 upon addition of azide in D85N and D85,96N mutants, whereas D85T shows no azide effect. The direction of these currents is the same as in wild type in yellow light. Yellow light alone is not sufficient to generate stationary currents in the mutants, but increasing yellow light intensity in the presence of blue light leads to an inversion of the current. Because all currents are carried by protons, this two-photon process demonstrates an inverted proton translocation by BR mutants.

Bacteriorhodopsins↗

PubMed1994

pH-induced structural changes in bacteriorhodopsin studied by Fourier transform infrared spectroscopy.

Previous C13-NMR studies showed that two of the four internal aspartic acid residues (Asp-96 and Asp-115) of bacteriorhodopsin (bR) are protonated up to pH = 10, but no accurate pKa of these residues has been determined. In this work, infrared spectroscopy with the attenuated total reflection technique was used to characterize pH-dependent structural changes of ground-state, dark-adapted wild-type bacteriorhodopsin and its mutant (D96N) with aspartic acid-96 replaced by asparagine. Data indicated deprotonation of Asp-96 at high pH (pKa = 11.4 +/- 0.1), but no Asp-115 titration was observed. The analysis of the whole spectral region characteristic to complex conformational changes in the protein showed a more complicated titration with an additional pKa value (pKa1 = 9.3 +/- 0.3 and pKa2 = 11.5 +/- 0.2). Comparison of results obtained for bR and the D96N mutant of bR shows that the pKa approximately 11.5 characterizes not a direct titration of Asp-96 but a protein conformational change that makes Asp-96 accessible to the external medium.

Aspartic Acid↗

PubMed1994

The flagellar bundle of Halobacterium salinarium is inserted into a distinct polar cap structure.

Flagellated envelopes of Halobacterium salinarium cells were prepared by lysis with taurodeoxycholate. After solubilization of the envelopes with Triton X-100 at high ionic strength, flagella and round patches from which numerous flagella emerged were isolated by gel filtration chromatography. We conclude that the flagellar bundle of H. salinarium is inserted into a differentiated polar cap structure.

Cell Fractionation↗

PubMed1994

Purification and characterization of a membrane-bound ATPase from Acetabularia cliftonii that corresponds to a Cl(-)-translocating ATPase in Acetabularia acetabulum.

A Mg(2+)-ATPase was solubilized from membranes of Acetabularia cliftonii using nonanoyl-N-methylgluconamide and purified by ion-exchange and gel permeation chromatography. One active ATPase fraction after Mono Q chromatography had a specific activity of 10 units/mg of protein. Judged from subunit composition [54 (a), 50 (b) with a fainter band around 40 kDa], catalytic properties, and N-terminal amino acid sequence of the b subunit, the isolated enzyme was comparable to the Cl(-)-ATPase of Acetabularia acetabulum. Immunological characterization of both subunits showed significant similarity to the F type of ATPase. Cl(-)-transport activity was observed by reconstitution studies into liposomes.

Acetabularia↗