Biomedical subjects

J Soppa

Publications and source records attributed to J Soppa.

15 recordsLinked to original sources

PubMed1998

A 71-kDa protein from Halobacterium salinarium belongs to a ubiquitous P-loop ATPase superfamily with head-rod-tail structure.

The nucleotide sequence of a genomic fragment from Halobacterium salinarium containing an open reading frame encoding a protein with a calculated molecular mass of 71 kDa was determined. Database searches revealed that this protein, Hp71, has similarities to eukaryotic cytoskeletal proteins. Heterologous production of Hp71 in Escherichia coli allowed the isolation of anti-Hp71 antibodies. The antibodies were used (1) to verify the production of Hp71 in H. salinarium and (2) to determine its cytoplasmic localization by immune electron microscopy. Homologous overproduction of Hp71 in H. salinarium and heterologous production in Haloferax volcanii resulted in modifications of cell morphology from rods to extended rods, and from pleiomorphic cells to rods, respectively. Structure prediction methods indicated that Hp71 has a head-rod-tail configuration, including an N-terminal domain with a nucleotide binding motif (P-loop), and an extended discontinuous coiled-coil domain of 330 amino acids. To identify related proteins, the complete genomes of Haemophilus influenzae, Mycoplasma genitalium, and Methanococcus jannaschii were searched for deduced proteins with extended coiled-coil domains. Only one or two proteins were found for each organism, showing that Hp71 is one of only a few prokaryotic intracellular proteins with extended coiled-coil domains. The phenotype upon overproduction and the similarity of Hp71 to the SMC superfamily of P-loop head-rod-tail proteins (named after SMC1, which is involved in the "stability of minichromosomes" in yeast) indicate that Hp71 might be involved in cytoskeleton formation and/or chromosome partitioning in H. salinarium.

Adenosine Triphosphatases↗

PubMed1997

The TATA-box-binding protein (TBP) of Halobacterium salinarum. Cloning of the tbp gene, heterologous production of TBP and folding of TBP into a native conformation.

The TATA-box binding protein (TBP) is a basal transcription factor involved in transcription initiation in Eucarya and Archaea. Using a tbp-specific probe, a 4.5-kbp genomic fragment from Halobacterium salinarum was cloned and sequenced. It contained the tbp gene and the 5'-ends of two additional open reading frames, but surprisingly, 70% of the cloned fragment (3.2 kbp) was devoid of coding capacity or similarity to database sequences. The deduced halobacterial TBP exhibits sequence similarities to other archaeal (41-43%) as well as to eucaryal (27-38%) TBP. A comparative analysis showed that the archaeal and eucaryal TBP form two related monophylic protein families, and the archaeal TBP possess features which separate them from eucaryal TBP. Compared with the other TBP, the halobacterial TBP is unique in having a high excess of negatively charged residues. A histidine-tagged version of the halobacterial TBP was produced in Escherichia coli in a denatured conformation and purified by means of Ni-chelating chromatography. CD spectroscopy was used to monitor TBP secondary structure and the conditions necessary for folding it into a native conformation. In the absence of denaturating agents, the folded as well as the unfolded state were found to be stable over a wide range of salt concentrations. Properly folded TBP was shown to bind to a halobacterial TATA-box-containing DNA fragment, indicating that the fusion protein can be used to characterize DNA recognition by the halobacterial TBP.

Amino Acid Sequence↗

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↗

PubMed1996

Characterization of the distal promoter element of halobacteria in vivo using saturation mutagenesis and selection.

The sequence and spacing requirements of the archaeal "distal promoter element' (DPE) were examined by randomizing positions -19 to -32 upstream of the transcriptional start site of the ferredoxin (fdx) promoter of Halobacterium salinarium. This randomized promoter library containing 4(14) entries was cloned in front of the dihydrofolate reductase (DHFR) reporter gene and transformed into Haloferax volcanii. Two approaches were used to characterize these synthetic promoters. First, 1040 independent clones were randomly chosen and their degrees of trimethoprim resistance were determined. The sequences of 20 clones that were either sensitive, partially resistant or very resistant, respectively, were determined. Secondly, the transformed library was screened by direct selection for high-activity promoters by growing transformants in the presence of trimethoprim. Both approaches produced the following consensus sequence for a halobacterial promoter: (Formula: see text) (where R = A or G; Y = C or T; W = A or T; S = G or C; N = A, C, G or T). Further characterization of two sensitive, two partially resistant, and two very resistant clones verified that DHFR activity and cell phenotype are directly correlated. Sensitive clones did not contain detectable dhfr mRNA, whereas partially resistant clones contained a 700 nucleotide (nt)-long transcript, and very resistant clones contained both the 700nt-long transcript and a second, more abundant, 500nt-long truncated transcript. Quantification of the dhfr mRNA and DHFR enzyme activity suggests that the 3'-untranslated region of the dhfr transcript, missing from the shorter transcript, functions as a negative regulator of translation.

Base Sequence↗

PubMed1996

Fermentative arginine degradation in Halobacterium salinarium (formerly Halobacterium halobium): genes, gene products, and transcripts of the arcRACB gene cluster.

Fermentative growth via the arginine deiminase pathway is mediated by the enzymes arginine deiminase, carbamate kinase, and catabolic ornithine transcarbamylase and by a membrane-bound arginine-ornithine antiporter. Recently we reported the characterization of catabolic ornithine transcarbamylase and the corresponding gene, arcB, from Halobacterium salinarium (formerly Halobacterium halobium). Upstream of the arcB gene, three additional open reading frames with halobacterial codon usage were found. They were identified as the arcC gene coding for carbamate kinase, the arcA gene coding for arginine deiminase, and a gene, tentatively termed arcR, coding for a putative regulatory protein. The identification of the arcC and arcA genes was verified, respectively, by heterologous expression of the enzyme in Haloferax volcanii and by protein isolation and N-terminal sequence determination of three peptides. The gene order arcRACB differs from the gene order arcDABC in Pseudomonas aeruginosa, the only other organism for which sequence information is available. Transcripts from H. salinarium cultures grown fermentatively or aerobically were characterized by Northern (RNA) blot and primer extension analyses. It was determined (i) that monocistronic transcripts corresponding to the four open reading frames exist and that there are three polycistronic transcripts, (ii) that the level of induction during fermentative growth differs for the various transcripts, and (iii) that upstream of the putative transcriptional start sites for the three structural genes there are sequences with similarities to the halobacterial consensus promoter. The data indicate that expression of the arc gene cluster and its regulation differ in H. salinarium and P. aeruginosa.

Amino Acid Sequence↗

PubMed1995

Catabolic ornithine transcarbamylase of Halobacterium halobium (salinarium): purification, characterization, sequence determination, and evolution.

Halobacterium halobium (salinarium) is able to grow fermentatively via the arginine deiminase pathway, which is mediated by three enzymes and one membrane-bound arginine-ornithine antiporter. One of the enzymes, catabolic ornithine transcarbamylase (cOTCase), was purified from fermentatively grown cultures by gel filtration and ammonium sulfate-mediated hydrophobic chromatography. It consists of a single type of subunit with an apparent molecular mass of 41 kDa. As is common for proteins of halophilic Archaea, the cOTCase is unstable below 1 M salt. In contrast to the cOTCase from Pseudomonas aeruginosa, the halophilic enzyme exhibits Michaelis-Menten kinetics with both carbamylphosphate and ornithine as substrates with Km values of 0.4 and 8 mM, respectively. The N-terminal sequences of the protein and four peptides were determined, comprising about 30% of the polypeptide. The sequence information was used to clone and sequence the corresponding gene, argB. It codes for a polypeptide of 295 amino acids with a calculated molecular mass of 32 kDa and an amino acid composition which is typical of halophilic proteins. The native molecular mass was determined to be 200 kDa, and therefore the cOTCase is a hexamer of identical subunits. The deduced protein sequence was compared to the cOTCase of P. aeruginosa and 14 anabolic OTCases, and a phylogenetic tree was constructed. The halobacterial cOTCase is more distantly related to the cOTCase than to the anabolic OTCase of P. aeruginosa. It is found in a group with the anabolic OTCases of Bacillus subtilis, P. aeruginosa, and Mycobacterium bovis.

Allosteric Regulation↗

PubMed1994

Two hypotheses--one answer. Sequence comparison does not support an evolutionary link between halobacterial retinal proteins including bacteriorhodopsin and eukaryotic G-protein-coupled receptors.

The structure of bacteriorhodopsin (BR) of Halobacterium halobium is known. Despite the lack of sequence similarities it is often taken as a model for eukaryotic G-protein-coupled receptors (GPCRs). Recently two hypotheses were used to support the homology of BR and GPCRs, namely evolution by exon shuffling and evolution by gene duplication. BR is a member of a family of halobacterial retinal proteins. The sequences of eight members of this family were used to test the two hypotheses. Based on sequence comparison, no indication for an evolutionary linkage between the two protein families could be found.

Amino Acid Sequence↗

PubMed1993

Bacterioopsin, haloopsin, and sensory opsin I of the halobacterial isolate Halobacterium sp. strain SG1: three new members of a growing family.

The genes coding for bacterioopsin, haloopsin, and sensory opsin I of a halobacterial isolate from the Red Sea called Halobacterium sp. strain SG1 have been cloned and sequenced. The deduced protein sequences were aligned to the previously known halobacterial retinal proteins. The addition of these new sequences lowered the number of conserved residues to only 23 amino acids, or 8% of the alignment. Data base searches with two highly conserved peptides as well as with an alignment profile yielded no significant similarity to any other protein, so the halobacterial retinal proteins should be regarded as a distinct protein family. The protein alignment was used to make predictions about the structure of the retinal proteins as well as about the amino acids in contact with retinal proteins. These results were in excellent agreement with the structural model of bacteriorhodopsin of Halobacterium halobium as well as with mutant studies, indicating that (i) structure predictions based on the sequences of a membrane protein family can be quite accurate; (ii) halorhodopsin and sensory rhodopsin I have tertiary structures similar to that of bacteriorhodopsin; (iii) conserved amino acids do not take part in reactions specific for one group of proteins, e.g., proton translocation for bacteriorhodopsins, but have a crucial role in determining the conformation and reactions of the chromophore; and (iv) the general mode of action (light-induced chromophore and protein movements) is the same for all halobacterial retinal proteins, ion pumps as well as sensors.

Amino Acid Sequence↗

PubMed1990

High resolution 13C-solid state NMR of bacteriorhodopsin: assignment of specific aspartic acids and structural implications of single site mutations.

Three mutant strains of Halobacterium sp. GRB with the site of mutation in the bacterioopsin gene (PM 326: Asp96----Asn; PM 374: Asp96----Gly; PM 384: Asp85----Glu) were grown in a synthetic medium containing (4-13C)-Asp. The mutant bacteriorhodopsins labeled with (4-13C)-Asp (37%-45%), and owing to the metabolism of Halobacteria also with (11-13C)-Trp (50%-100%), were isolated as purple membranes and 13C Solid State Magic Angle Sample Spinning (MASS) Nuclear Magnetic Resonance (NMR) spectra of the samples were taken. The Asp96 mutants lacked the signal at 171.3 ppm which was previously assigned to a protonated internal Asp (Engelhard et al. 1989a). This observation supports the conclusion that Asp96 is protonated in the ground state. PM 384 (Asp85----Glu) has an absorption maximum at 610 nm. It can be converted into a purple form (lambda max = 540 nm) by treatment with a detergent (CHAPSO). The NMR-spectra of these two species differ from each other and from the wild type. The intensity of the resonance at 173 ppm in the wild type spectrum is reduced in both forms of the mutant protein. It is probable that this signal is caused by Asp85. The amino acid changes result not only in a perturbation of their direct environment but also effects on Trp residues and the chromophore protein interaction can be observed.

Aspartic Acid↗

PubMed1989

Bacteriorhodopsin mutants of Halobacterium sp. GRB. II. Characterization of mutants.

The bacterioopsin genes of Halobacterium sp. GRB (Ebert, K., Goebel, W., and Pfeifer, F. (1984) Mol. & Gen. Genet. 194, 91-97) wild type and 10 independent mutants of different phenotypes have been cloned and sequenced. The wild type gene has two conservative changes compared to the gene of Halobacterium halobium, so that the proteins of the two species are identical. Six different mutations at five different codons have been found, leading to the following amino acid changes compared to the wild type: Trp10----Cys (three cases), Tyr57----Asn, Asp85----Glu, Asp06----Asn (three cases), Asp96----Gly, Trp138----Arg. A first characterization of the mutant proteins is given, and their implications for models of bacteriorhodopsin structure and function are discussed.

Amino Acid Sequence↗

PubMed1989

Bacteriorhodopsin mutants of Halobacterium sp. GRB. I. The 5-bromo-2'-deoxyuridine selection as a method to isolate point mutants in halobacteria.

Halobacterium sp. GRB (Ebert, K., Goebel, W., and Pfeifer, F. (1984) Mol. & Gen. Genet. 194, 91-97) was used to isolate bacteriorhodopsin (BR) mutants. A procedure is described which allows the enrichment of any type of mutant unable to grow under the selection conditions applied. Its use for the isolation of phototrophically negative, retinal-positive mutants of Halobacterium sp. GRB is demonstrated. Single-cell clones of this phenotype were further characterized. The expression of bacterioopsin was tested with a monoclonal antibody directed against the C terminus of the protein. The expressed bacteriorhodopsins were characterized by their specific activity for proton pumping, their spectral properties, and photocycle kinetics. About 15 independent mutants carrying bacteriorhodopsins of three distinct phenotypic classes could be isolated, including BR with a different absorption maximum, BR of lower specific activity, and BR characterized by a slower photocycle and a lack of proton pumping activity.

Bacteriorhodopsins↗

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

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↗

PubMed1987

Lactose permease of Escherichia coli: properties of mutants defective in substrate translocation.

Mutants of lactose permease of Escherichia coli with amino acid changes (Gly-24----Glu; Gly-24----Arg; Pro-28---Ser; Gly-24, Pro-28----Glu-Ser and Gly-24, Pro-28----Arg-Ser) within a putative membrane-spanning alpha-helix (Phe-Gly-Leu-Phe-Phe-Phe-Phe-Tyr-Phe-Phe-Ile-Met-Gly- Ala-Tyr-Phe-Pro-Phe-Phe-Pro-Ile) are incorporated into the cytoplasmic membrane. The mutant proteins retain the ability to bind galactosides, and the affinity for several substrates is actually increased. However, the rate of active transport is decreased to 0.01% of the wild-type rate in the mutants carrying Arg-24 or Arg-24, Ser-28. Kinetic analysis demonstrates that the two mutants require 10 min to cause occupied binding sites for galactoside and H+ to change their exposure from the periplasm to the cytoplasm as compared to 50 ms in the wild type. The effect is less pronounced when these sites are unoccupied.

Amino Acid Sequence↗

PubMed1985

Properties of a mutant lactose carrier of Escherichia coli with a Cys148----Ser148 substitution.

The cysteine residue at position 148 in the lactose carrier protein of Escherichia coli has been replaced by serine using oligonucleotide-directed, site-specific mutagenesis of the lac Y gene. The mutant carrier is incorporated into the cytoplasmic membrane to the same extent as the wild-type carrier, confers a lactose-positive phenotype on cells, and actively transports lactose and other galactosides. However, the maximum rate of transport for several substrates is reduced by a factor of 6-10 while the apparent affinity is reduced by a factor of 2-4. Carrier activity in the mutant is much less sensitive to sulfhydryl reagents (HgCl2, p-(chloromercuri)benzenesulfonate and N-ethylmaleimide) than in the wild type, and beta-D-galactosyl 1-thio-beta-D-galactoside does not protect the mutant carrier against slow inactivation by N-ethylmaleimide. It is concluded that the Cys148 residue is not essential for carrier-catalyzed galactoside: proton symport and that its alkylation presumbly prohibits access of the substrate to the binding site by steric hindrance. A serine residue at position 148 in the amino acid sequence appears to alter the protein structure in such a way that one or more sulfhydryl groups elsewhere in the protein become accessible to alkylating agents thereby inhibiting transport. Recently, Trumble et al. [(1984) Biochem. Biophys. Res. Commun. 119, 860-867] arrived at similar conclusions by investigating a mutant carrier with a Cys148----Gly148 replacement.

Amino Acid Sequence↗