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Biomedical subjects

D Oesterhelt

Publications and source records attributed to D Oesterhelt.

At least 163 records · Page 9Linked to original sources
PubMed1989

A defective proton pump, point-mutated bacteriorhodopsin Asp96----Asn is fully reactivated by azide.

Addition of azide fully restored the proton pump activity of defective bacteriorhodopsin (BR) mutant protein Asp96----Asn. The decay time of M of BR Asp96----Asn, the longest living intermediate, was decreased from 500 ms at pH 7.0 to approximately 1 ms under conditions of saturating azide concentrations. This decay was faster than the decay of M in the wild-type, where no such azide effect was detectable. Stationary photocurrents, measured with purple membranes immobilized and oriented in a polyacrylamide gel, increased upon addition of azide up to the level of the wild-type. Different small anions of weak acids restored the pump activity with decreasing affinity in the order: cyanate greater than azide greater than nitrite greater than formiate greater than acetate. The activation energy of the M decay in the mutant was higher in the presence (48 kJ/mol) than in the absence (27 kJ/mol) of 100 mM azide even though the absolute rate was dramatically increased by azide. This effect of azide is due to the substitution of a carboxamido group for a carboxylic group at position 96 which removes the internal proton donor and causes an increase in the entropy change of activation for proton transfer which is reversed by azide.

Anions↗
PubMed1989

Cloning and sequencing of the fbcF, B and C genes encoding the cytochrome b/c1 complex from Rhodopseudomonas viridis.

The complete nucleotide sequence of the genes encoding the Rieske FeS, the cytochrome b and the cytochrome c1 subunits of the ubiquinol-cytochrome c2 oxidoreductase from the photosynthetic purple bacterium Rhodopseudomonas viridis, and the derived amino acid sequences are presented. These three genes, fbcF, fbcB and fbcC, are located at contiguous sites of the genome. The DNA-deduced amino acid sequences are compared with known primary structures of corresponding proteins from other purple photosynthetic bacteria, as well as mitochondria, cyanobacteria and chloroplasts.

Amino Acid Sequence↗
PubMed1989

Two pumps, one principle: light-driven ion transport in halobacteria.

Comparison of the primary structure of the chloride pump halorhodopsin with that of the proton pump bacteriorhodopsin provides insight into light-driven ion transport by retinal proteins. Several conserved amino acid residues in the membrane-spanning region of both proteins and their interaction with different isomerization states of retinal are suggested to be the key element for ion transport in both proteins.

Amino Acid Sequence↗
PubMed1989

Role of aspartate-96 in proton translocation by bacteriorhodopsin.

Proton transfer reactions in bacteriorhodopsin were investigated by Fourier transform infrared spectroscopy, using a mutant protein in which Asp-96 was replaced by Asn-96. By comparison of the BR - K, BR - L, and BR - M difference spectra (BR indicating bacteriorhodopsin ground state and K, L, and M indicating photo-intermediates) of the wild-type protein with the corresponding difference spectra of the mutant protein, detailed insight into the functional role of this residue in the proton pump mechanism is obtained. Asp-96 is protonated in BR, as well as another aspartic residue, which is tentatively assigned to be Asp-115. Asp-96 is not affected in the primary photoreaction. During formation of the L intermediate it is subjected to a change in the H-bonding character of its carboxylic group, but no deprotonation occurs at this reaction step. Also, in the mutant protein a light-induced structural change of the protein interior near the Asn-96 residue is probed. The BR - M difference spectrum of the mutant protein lacks the negative carbonyl band at 1742 cm-1 of Asp-96 and in addition a positive band at about 1378 cm-1, which is most likely to be caused by the carboxylate vibration of Asp-96. This argues for a deprotonation of Asp-96 in the time range of the M intermediate during its photostationary accumulation. On the basis of these results, it is suggested that the point mutation does not induce a gross change of the protein structure, but a proton-binding site in the proton pathway from the cytoplasmic side to the Schiff base is lost.

Asparagine↗
PubMed1989

Biosynthesis of the two halobacterial light sensors P480 and sensory rhodopsin and variation in gain of their signal transduction chains.

The two retinal-containing photoreceptors of halobacteria, P480 and sensory rhodopsin, are formed constitutively and inducibly, respectively. Both photoreceptors are synthesized as apoproteins in cells with nicotine-inhibited retinal synthesis and are reconstituted as chromoproteins by the addition of all-trans retinal to cell membrane preparations. The decrease in photoreceptor-mediated photophobic response at the stationary growth phase of cells is not due to photoreceptor degradation but due to a deficiency of the signal transduction chain in the cell.

Bacterial Proteins↗
PubMed1989

Microaerophilic growth and induction of the photosynthetic reaction center in Rhodopseudomonas viridis.

Rhodopseudomonas viridis was grown in liquid culture at 30 degrees C anaerobically in light (generation time, 13 h) and under microaerophilic growth conditions in the dark (generation time, 24 h). The bacterium could be cloned at the same temperature anaerobically in light (1 week) and aerobically in the dark (3 to 4 weeks) if oxygen was limited to 0.1%. Oxygen could not be replaced by dimethyl sulfoxide, potassium nitrate, or sodium nitrite as a terminal electron acceptor. No growth was observed anaerobically in darkness or in the light when air was present. A variety of additional carbon sources were used to supplement the standard succinate medium, but enhanced stationary-phase cell density was observed only with glucose. Conditions for induction of the photosynthetic reaction center upon the change from microaerophilic to phototrophic growth conditions were investigated and optimized for a mutant functionally defective in phototrophic growth. R. viridis consumed about 20-fold its cell volume of oxygen per hour during respiration. The MICs of ampicillin, kanamycin, streptomycin, tetracycline, 1-methyl-3-nitro-1-nitrosoguanidine, and terbutryn were determined.

Bacterial Proteins↗
PubMed1989

Gene transfer system for Rhodopseudomonas viridis.

A gene transfer system for Rhodopseudomonas viridis was established which uses conjugation with Escherichia coli S17-I as the donor and mobilizable plasmids as vectors. Initially, plasmids of the incompatibility group P1 (pRK290 and pRK404) were used. The more effective shuttle vectors between E. coli and R. viridis, pKV1 and pKVS1, were derived from plasmid pBR322 and showed the highest conjugation frequency (10(-2] thus far demonstrated in purple bacteria. It was also demonstrated that Rhizobium meliloti can be used as a donor for conjugation with R. viridis. From a genomic cosmid library of R. viridis constructed in the vector pHC79, clones that coded for subunits H (puh operon), L, M and cytochrome c (puf operon) of the photosynthetic reaction center were isolated and characterized. For linkage of the two operons on the genome, cosmids that overlapped with the operon-carrying clones were identified. The relative positions of the two operons could not be determined, but the operons must be more than 100 kilobase pairs apart. Thus, the genomic organization of the reaction center in R. viridis is different from that of Rhodobacter capsulatus, for which a distance of about 39 kilobase pairs was determined. From a spontaneous mutant of R. viridis that is resistant to the herbicide terbutryn, the puf operon was cloned in pKVS1 and transferred by conjugation into R. viridis wild-type cells. The resulting exconjugants were resistant to the herbicide, which demonstrated that the puf operon on pKVS1 constructions was functionally expressed in R. viridis.

Cloning, Molecular↗
PubMed1989

Halobacterium sp. GRB: a species to work with!?

The properties of the halobacterial isolate Halobacterium sp. GRB are discussed, especially in relation to its use as a laboratory strain. Experimental results on this species are described, including the isolation of point mutants in the bacterioopsin gene leading to single amino acid replacements in bacteriorhodopsin, the application of a selection procedure for the isolation of different types of mutants, the genetic stability of Halobacterium sp. GRB and the possibility of isolating a set of isogenic mutants, the conditions for transformation experiments with this species, and specific features of Halobacterium sp. GRB, such as halocin production and the absence of a restriction system, as well as DNA adenosine methylation.

Bacteriorhodopsins↗
PubMed1988

Cloning and determination of the nucleotide sequence of the Mn-containing superoxide dismutase gene from Halobacterium halobium.

A group of synthetic 17-mer oligodeoxynucleotides (oligos) was constructed to correspond to a sequence of amino acids situated near the N terminus of the manganese-containing superoxide dismutase (Mn-SOD) purified from the halophilic bacterium, Halobacterium halobium. A cosmid library of a Sau3AI partial digest of halobium DNA, cloned into the BamHI site of pHC79, was probed with the radiolabeled oligos. Cosmid DNA was purified from the clone that showed hybridization at the highest stringency. A 1.8-kb PstI fragment of this DNA which hybridized the probes was subcloned into bacteriophage M13 and transfected into Escherichia coli JM101. The entire insert containing a 600-bp sequence coding for Mn-SOD and its 5'- and 3'-flanking regions was sequenced. The derived amino acid sequence of the structural gene showed a similarity to other manganese and iron-containing SODs in normally conserved regions.

Amino Acid Sequence↗
PubMed1988

Methoxyretinals in bacteriorhodopsin. Absorption maxima, cis-trans isomerization and retinal protein interaction.

Analogue bacteriorhodopsins (BRs) were reconstituted from bacterioopsin and 9-, 11-, or 13-methoxyretinals or their demethyl derivatives, respectively. In organic solvents the retinals occur as cis isomers of the respective double bonds carrying the methoxy group. 9-Methoxyretinal, present as the 9-cis isomer, does not form an analogue BR with bacterioopsin in the dark. Upon illumination, a BR is produced with an absorbance maximum at 560 nm. This compound is thermally unstable, and converts back into the 9-cis-containing complex (lambda max = 410 nm) in the dark. Removal of the 13-methyl group from this compound (= 9-methoxy 13-demethyl retinal) does not change the 9-cis configuration of the free retinal, but allows the reconstitution of a thermally stable chromoprotein absorbing around 500 nm with a proton translocation rate of about 10% of the BR value, comparable to the 13-demethyl BR value [Gärtner, W., Towner, P., Hopf, H. & Oesterhelt, D. (1983) Biochemistry 22, 2637-2644]. 11-Methoxy BRs (13-demethyl and 9,13-didemethyl) absorb around 530 nm and are inactive. 13-Methoxy retinal (13-cis isomer) reconstitutes a chromoprotein with an absorbance maximum at 515 nm, which can be photoconverted to a thermostable 460-nm-absorbing complex. For the 515-nm-absorbing species of 13-methoxy BR a light-induced proton translocation was not detected in measurements with cell vesicles (detection of pH changes in the vesicle preparation). Only by photocurrent measurements in a bilayer experiment could a very diminished photocurrent be detected, about 1-2% of BR, [Fendler et al. (1987) Biochim. Biophys. Acta 893, 60-68]. The reconstitution rate of 13-methoxy BR from 13-methoxy retinal and bacterioopsin is slower by a factor of 40 compared to 13-ethyl BR, although both substituents are of similar size. The position 13 of retinal was found to be most sensitive for regulation of the absorption maximum and the formation and stability of the all-trans isomer, which is the active form for light-induced proton translocation. The results suggest that an electronic interaction with a charged residue of the binding site exists around position 13 of retinal, which is disturbed when a methoxy group replaces the methyl or ethyl group at that position. This electronic interaction is essential for maintaining the active all-trans configuration of retinal.

Bacteriorhodopsins↗
PubMed1988

The orientation of halorhodopsin in the cell membrane of halobacteria.

The orientation of the light-driven chloride pump, halorhodopsin, in the membrane was determined using antibodies directed against a synthetic peptide which represents the C-terminal segment of the protein. Antibodies against this decapeptide did not bind to right-side-out cell vesicles. Partial inversion by sonication or lysis under low salt conditions exposed this COOH-terminal antigenic site. Antibody binding was removed by preincubation with the decapeptide. The COOH terminus of the molecule is therefore located on the cytoplasmic surface of the membrane.

Amino Acid Sequence↗
PubMed1988

The sodium ion translocating oxalacetate decarboxylase of Klebsiella pneumoniae. Sequence of the biotin-containing alpha-subunit and relationship to other biotin-containing enzymes.

The gene encoding the alpha-subunit of the Na+ pump oxalacetate decarboxylase of Klebsiella pneumoniae was cloned and sequenced. The deduced primary structure of the protein was confirmed by protein sequencing of about 30% of the polypeptide chain. The gene has a GC content of 67% and codes for 596 amino acids. The N-terminal methionine is removed in the mature protein which has a calculated molecular mass of 63,600 daltons. The protein consists of two different domains that are connected by a stretch of amino acid residues susceptible to proteolytic cleavage. Limited proteolysis of the native enzyme with trypsin produced fragments of about 51 kDa and 10.2 kDa, the latter of which started with valine 491 and contained the biotin prosthetic group. Peptide sequencing indicated binding of the biotin prosthetic group to lysine 561, 35 residues from the C terminus. The decarboxylase contains an extended alanine- and proline-rich region (positions 502-532) on the N-terminal side of the 10.2-kDa biotin domain. This sequence includes a total of 16 alanine and 9 proline residues.

Alanine↗
PubMed1988

Purification of a manganese-containing superoxide dismutase from Halobacterium halobium.

An oxygen-induced superoxide dismutase was purified from the halophilic bacterium, Halobacterium halobium, strain NRL. Due to the high salt requirement for enzyme stability, the purification had to be performed in the presence of 2 M NaCl. The pI of the protein was 4.95. The approximate Mr was 38,500. The subunit size as determined by sodium dodecyl sulfate-electrophoresis was approximately 19,000. Metal analysis showed 1.5 atoms of manganese per dimer, 0.5 atom zinc, and 1.54 atoms copper. The N-terminal sequence of amino acids was determined, and based upon the first 26 amino acids significant homology to other manganese- and iron-containing superoxide dismutases was revealed.

Amino Acid Sequence↗
PubMed1988

Structure and orientation of halorhodopsin in the membrane: a proteolytic fragmentation study.

Halorhodopsin (HR), the light-driven chloride pump in halobacteria, was digested with various proteolytic enzymes. As expected, carboxypeptidase A removed 14 amino acids from the C-terminal tail of detergent-solubilized HR, producing a fragment of 25.2 kd in size. Membrane-associated HR could be digested as well, but not in right-side-out sealed cell envelope vesicles. We conclude, therefore, that the orientation of HR in the cytoplasmic membrane is such that the C-terminal tail faces the cytoplasmic side. Tryptic digestion of detergent-solubilized HR resulted in the removal of the same C-terminal segment, but also in the production of two more cleavage products (molecular masses of 20.9 and 16.8 kd respectively). These cleavage sites were determined by amino acid sequencing of the newly produced N termini, and they turned out to be within interhelical loops in an earlier proposed structural model for HR. Incubation with chymotrypsin and thermolysin yielded different sites of cleavage, but also in regions which were proposed to be accessible on the surface of the protein. Since the results show that three of six proposed interhelical loop segments contain proteolytic digestion sites, they support the proposed structural model for HR.

Amino Acid Sequence↗
PubMed1988

Isolation of a prokaryotic photoreceptor: sensory rhodopsin from halobacteria.

The photoreceptor sensory rhodopsin was isolated from halobacterial cell membranes solubilized in laurylmaltoside. In the presence of retinal, detergent and salt the native protein was obtained in pure form by sucrose density gradient centrifugation, hydroxyapatite chromatography and gel filtration. The apparent mol. wt of the molecule was 24 kd if analyzed by SDS gel electrophoresis, and 49 kd by sedimentation and size-exclusion chromatographic analysis. The chromoprotein had an absorption maximum at 580 nm which was 8 nm blue-shifted compared to the membrane-bound state. The molecule was photochemically active and the action spectrum for formation of SR380, the long-lived intermediate, coincided with the absorption spectrum.

Centrifugation, Density Gradient↗
PubMed1988

Lysis of halobacteria in bacto-peptone by bile acids.

All tested strains of halophilic archaebacteria of the genera Halobacterium, Haloarcula, Haloferax, and Natronobacterium lysed in 1% Bacto-Peptone (Difco) containing 25% NaCl, whereas no lysis was observed with other strains belonging to archaebacteria of the genera Halococcus, Natronococcus, and Sulfolobus, methanogenic bacteria, and moderately halophilic eubacteria. Substances in Bacto-Peptone which caused lysis of halobacteria were purified and identified as taurocholic acid and glycocholic acid. High-performance liquid chromatography analyses of peptones revealed that Bacto-Peptone contained nine different bile acids, with a total content of 9.53 mg/g, whereas much lower amounts were found in Peptone Bacteriological Technical (Difco) and Oxoid Peptone. Different kinds of peptones can be used to distinguish halophilic eubacteria and archaebacteria in mixed cultures from hypersaline environments.

Journal Article↗