Biomedical subjects

R Sterner

Publications and source records attributed to R Sterner.

At least 37 records · Page 2Linked to original sources

PubMed1996

Phosphoribosyl anthranilate isomerase from Thermotoga maritima is an extremely stable and active homodimer.

The metabolism of hyperthermophilic microorganisms can function properly at temperatures close to 100 degrees C. It follows that they are equipped with both thermostable enzymes and mechanisms that handle labile metabolites. We wanted to understand how stable and active phosphoribosyl anthranilate isomerase (tPRAI) from the hyperthermophile Thermotoga maritima is at its optimum growth temperature of 80 degrees C, and how its thermolabile substrate, N-(5'-phosphoribosyl)-anthranilate (PRA), is protected from rapid decomposition. To this end, the trpF gene of T. maritima was expressed heterologously in Escherichia coli and tPRAI was purified. In contrast to most PRAIs from mesophiles, which are monomers with the eightfold beta alpha (or TIM) barrel fold, tPRAI is a homodimer. It is strongly resistant toward inactivation by temperatures up to 95 degrees C, by acidification to pH 3.2, and by proteases in the presence and absence of detergents. tPRAI is about 35-fold more active at its physiologic temperature than is the enzyme from E. coli (ePRAI) at 37 degrees C. This high catalytic efficiency of tPRAI is likely to complete successfully with the rapid spontaneous hydrolysis of PRA at 80 degrees C. Thus, with respect to both stability and function, tPRAI appears well adapted to the extreme habitat of T. maritima. Single crystals of tPRAI have been obtained that are suitable for X-ray analysis at high resolution.

Aldose-Ketose Isomerases↗

PubMed1995

2.0 A structure of indole-3-glycerol phosphate synthase from the hyperthermophile Sulfolobus solfataricus: possible determinants of protein stability.

BACKGROUND: Recent efforts to understand the basis of protein stability have focused attention on comparative studies of proteins from hyperthermophilic and mesophilic organisms. Most work to date has been on either oligomeric enzymes or monomers comprising more than one domain. Such studies are hampered by the need to distinguish between stabilizing interactions acting between subunits or domains from those acting within domains. In order to simplify the search for determinants of protein stability we have chosen to study the monomeric enzyme indole-3-glycerol phosphate synthase from the hyperthermophilic archaeon Sulfolobus solfataricus (sIGPS), which grows optimally at 90 degrees C. RESULTS: The 2.0 A crystal structure of sIGPS was determined and compared with the known 2.0 A structure of the IGPS domain of the bifunctional enzyme from the mesophilic bacterium Escherichia coli (eIGPS). sIGPS and eIGPS have only 30% sequence identity, but share high structural similarity. Both are single-domain (beta/alpha)8 barrel proteins, with one (eIGPS) or two (sIGPS) additional helices inserted before the first beta strand. The thermostable sIGPS has many more salt bridges than eIGPS. Several salt bridges crosslink adjacent alpha helices or participate in triple or quadruple salt-bridge clusters. The number of helix capping, dipole stabilizing and hydrophobic interactions is also increased in sIGPS. CONCLUSIONS: The higher stability of sIGPS compared with eIGPS seems to be the result of several improved interactions. These include a larger number of salt bridges, stabilization of alpha helices and strengthening of both polypeptide chain termini and solvent-exposed loops.

Aldehyde Oxidoreductases↗

PubMed1995

(Beta alpha)8-barrel proteins of tryptophan biosynthesis in the hyperthermophile Thermotoga maritima.

To better understand the evolution of a key metabolic pathway, we have sequenced the trpCFBA gene cluster of the hyperthermophilic bacterium Thermotoga maritima. The genes were cloned by complementation in vivo of trp deletion strains of Escherichia coli. The new sequences, together with earlier findings, establish that the trp operon of T.maritima has the order trpE(G.D)CFBA, which might represent the ancestral organization of the tryptophan operon. Heterologous expression of the trp(G.D) and trpC genes in E.coli and N-terminal sequencing of their polypeptide products showed that their translation is initiated at the rate start codons TTG and ATC, respectively. Consequently, the N-terminus of the trp(G.D) fusion protein is 43 residues shorter than previously postulated. Amino acid composition and sequence analyses of the protein products of T.maritima trpC (indoleglycerol phosphate synthase), trpF (phosphoribosyl anthranilate isomerase) and trpA (alpha-subunit of tryptophan synthase) suggest that these thermostable (beta alpha)8-barrel proteins may be stabilized by additional salt bridges, compared with the mesostable forms. Another notable feature is the predicted lack of the N-terminal helix alpha 0 in the alpha-subunit of tryptophan synthase.

Aldose-Ketose Isomerases↗

PubMed1995

Extreme thermostability of tarantula hemocyanin.

Biotops with extreme temperatures such as deserts force animals to avoid or escape high temperatures by biochemical, behavioural or morphological adaptation. In this context we tested the resistance to heat of the oxygen carrier hemocyanin from the ancient tarantula Eurypelma californicum, which is found in arid zones of North America. Differential scanning calorimetry, light scattering, crossed immunogelelectrophoresis and oxygen binding experiments show that the 24-meric hemocyanin is conformationally stable and fully functioning at temperatures up to 90 degrees C. Our results demonstrate that the cation-mediated state of oligomerization is not only crucial for the high cooperativity of oxygen binding of this hemocyanin, but also for its extreme stability in the physiological temperature and pH range.

Adaptation, Physiological↗

PubMed1995

1H nuclear-magnetic-resonance investigation of oxidized Fe4S4 ferredoxin from Thermotoga maritima. Hyperfine-shifted resonances, sequence-specific assignments and secondary structure.

The oxidized Fe4S4 ferredoxin from the hyperthermophilic bacterium Thermotoga maritima has been investigated by one- and two-dimensional NMR in order to characterize its hyperfine-shifted resonances originating from the cysteinyl cluster ligands and to assign its resonances in the diamagnetic shift range. The chemical shift and relaxation time pattern of the hyperfine-shifted signals is very similar to other oxidized Fe4S4 ferredoxins. A tentative sequence-specific assignment of these resonances according to a general pattern of chemical shift of cysteine protons versus sequence position of cluster ligand is presented. Furthermore, sequence-specific assignments for 85% of the amino acid residues that were obtained without any guidance by known X-ray structures of ferredoxins are given. They reveal the formation of at least two elements of secondary structure by the polypeptide chain of T. maritima ferredoxin: an alpha-helix comprising residues C43-D49 and a double-stranded antiparallel beta-sheet consisting of the N- and C-terminal parts of the protein. This folding pattern is very similar to that of the crystallographically characterized ferredoxin from the mesophile Desulfovibrio gigas [Kissinger, C.R., Sieker, L.C., Adman E.T. & Jensen, L.H. (1991) J. Mol. Biol. 219, 693-715] and therefore suggesting different mechanisms of stabilization for T. maritima ferredoxin and the ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus that was recently investigated by NMR [Teng, Q., Zhou, Z.H., Smith, E.T., Busse, S. C., Howard, J.B., Adams M.W.W. & La Mar, G.N. (1994) Biochemistry 33, 6316-6326].

Amino Acid Sequence↗

PubMed1994

Inversion of the Bohr effect upon oxygen binding to 24-meric tarantula hemocyanin.

The Bohr effect describes the usually negative coupling between the binding of oxygen and the binding of protons to respiratory proteins. It was first described for hemoglobin and provides for an optimal oxygen supply of the organism under changing physiological conditions. Our measurements of both oxygen and proton binding to the 24-meric tarantula hemocyanin establish the unusual case where a respiratory protein binds protons at low degrees of oxygenation but releases protons at high degrees of oxygenation. In contrast to what is observed with hemoglobin and other respiratory proteins, this phenomenon amounts to the inversion of the Bohr effect in the course of an oxygen-binding curve at a given pH value. Therefore, protons in spider blood can act either as allosteric activators or as allosteric inhibitors of oxygen binding, depending on the degree of oxygenation of hemocyanin. These functional properties of tarantula hemocyanin, which cannot be explained by classical allosteric models, require at least four different conformational states of the subunits. Inspection of the known x-ray structures of closely related hemocyanins suggests that salt bridges between completely conserved histidine and glutamate residues located at particular intersubunit interfaces are responsible for the observed phenomena.

Animals↗

PubMed1994

Sequence, assembly and evolution of a primordial ferredoxin from Thermotoga maritima.

A gene coding for the ferredoxin of the primordial, strictly anaerobic and hyperthermophilic bacterium Thermotoga maritima was cloned, sequenced and expressed in Escherichia coli. The ferredoxin gene encodes a polypeptide of 60 amino acids that incorporates a single 4Fe-4S cluster. T. maritima ferredoxin expressed in E. coli is a heat-stable, monomeric protein, the spectroscopic properties of which show that its 4Fe-4S cluster is correctly assembled within the mesophilic host, and that it remains stable during purification under aerobic conditions. Removal of the iron-sulfur cluster results in an apo-ferredoxin that has no detectable secondary structure. This observation indicates that in vivo formation of the ferredoxin structure is coupled to the insertion of the iron-sulfur cluster into the polypeptide chain. Sequence comparison of T. maritima ferredoxin with other 4Fe-4S ferredoxins revealed high sequence identities (75% and 50% respectively) to the ferredoxins from the hyperthermophilic members of the Archaea, Thermococcus litoralis and Pyrococcus furiosus. The high sequence similarity supports a close relationship between these extreme thermophilic organisms from different phylogenetic domains and suggests that ferredoxins with a single 4Fe-4S cluster are the primordial representatives of the whole protein family. This observation suggests a new model for the evolution of ferredoxins.

Amino Acid Sequence↗

PubMed1994

Tris: an allosteric effector of tarantula haemocyanin.

The effect of the chemical buffering component Tris (hydroxy-methyl-amino-methane) and of chloride ions on the oxygen binding of tarantula hemocyanin was studied at constant pH. It revealed that Tris at micromolar concentrations decreases the oxygen pressure at half-saturation (p50) by a factor of more than two, whereas chloride does not influence oxygen affinity. A thermodynamic analysis in terms of the nested model of allostery [(1987) Proc. Natl. Acad. Sci. 84, 1891-1895] indicated that Tris acts a an allosteric activator of oxygen binding by influencing the interaction between the 12-meric half-molecules of the 24-meric tarantula haemocyanin.

Allosteric Regulation↗

PubMed1990

Factors affecting nucleosome structure in transcriptionally active chromatin. Histone acetylation, nascent RNA and inhibitors of RNA synthesis.

The nucleosomes of transcriptionally active genes can be separated from those of inactive genes by affinity chromatography on organomercury-agarose (Hg-agarose) columns. The basis for this separation is the difference in accessibility of the sulfhydryl groups of histone H3 and certain non-histone proteins in active and inactive chromatin. A new procedure distinguishing between different modes of binding of transcriptionally active nucleosomes to the Hg-agarose column has been applied to study several factors which might influence the binding reaction. Nucleosomes that bind to the column because of salt-labile associations with SH-reactive non-histone proteins, such as the high-mobility-group proteins, HMG-1 and HMG-2, were released by adding 0.5 M NaCl to the eluting buffer. The remaining nucleosomes, in which reactive histone H3 thiol groups can bind covalently to the organomercury, were then displaced from the column by 10 mM dithiothreitol. Both Hg-agarose-bound fractions contain the transcriptionally active DNA sequences of the cell, but inactive nucleosomes, such as those containing alpha-globin DNA, pass through the column. The histones of both Hg-agarose-bound fractions have significantly higher levels of acetylation than do histones of the unbound fraction, but the content of tri- and tetra-acetylated H3 and H4 is significantly higher in the nucleosomes with reactive H3 thiols. The rate of turnover of histone N-acetyl groups is also far greater in the Hg-agarose-bound nucleosomes than in the unbound nucleosomes. Although the overall levels of histone acetylation can be increased significantly by incubating HeLa cells in the presence of the deacetylase inhibitor, 5 mM sodium butyrate, this treatment has little if any effect on the total number of nucleosomes retained on the Hg-agarose column. However, the ability of Hg-agarose chromatography to detect localized changes in chromatin structure is evidenced by an 11-fold increase in the Hg-agarose binding of nucleosomes containing the DNA of the butyrate-inducible alkaline phosphatase gene, compared to the Hg-agarose-bound nucleosomes of control cells. Although nascent RNA chains are present in the Hg-agarose-bound nucleosomes released by dithiothreitol, binding of the SH-reactive nucleosomes to the Hg-agarose column is not dependent on the presence of proteins associated with nascent RNA chains, since binding does not decrease following removal of the nascent transcripts by ribonuclease treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylation↗

PubMed1990

Reversible and irreversible changes in nucleosome structure along the c-fos and c-myc oncogenes following inhibition of transcription.

A new affinity chromatographic procedure for the separation of transcriptionally active nucleosomes has been used to study the changes that take place in chromatin structure along the c-fos and c-myc genes when RNA synthesis is inhibited. Mercury-affinity chromatography separates the sulfhydryl-reactive nucleosomes of transcriptionally active genes from the compactly beaded, non-reactive nucleosomes of transcriptionally inert DNA sequences. The new procedure also discriminates between nucleosomes that have "unfolded" to reveal the previously shielded SH groups of histone H3 and nucleosomes that bind to the mercury column because of their association with thiol-containing non-histone proteins located in the transcription unit. Both classes of Hg-bound nucleosomes contain the c-fos and c-myc sequences, but only when they are being transcribed. We compared the effects of alpha-amanitin and actinomycin D on the transcription of c-fos and c-myc with the effects of each inhibitor on the distribution of the corresponding oncogenic DNA sequences in the chromatographically separated nucleosome fractions. It was found that the inhibition of RNA polymerase II by alpha-amanitin (added at the peaks of c-fos or c-myc expression in serum-stimulated BALB/c 3T3 cells) resulted in a rapid loss of affinity of the oncogene-containing nucleosomes for the mercury column. There was no corresponding effect on the mercury-binding properties of nucleosomes containing 28 S ribosomal gene sequences, which continue to be transcribed by amanitin-resistant RNA polymerase I. Therefore, the binding of the c-fos and c-myc nucleosomes to the mercury column seems to depend upon reversible structural changes associated with their transcription. Surprisingly, there was no corresponding loss of affinity of the c-fos and c-myc nucleosomes for the mercury column when actinomycin D was employed to inhibit RNA synthesis, despite the fact that transcription of both genes had been arrested abruptly. Measurements of [3H]actinomycin D binding show its preferential intercalation into the transcriptionally active nucleosomes. We suggest that the intercalation of actinomycin D into the DNA of active nucleosomes can lock the transcription complex into an "unfolded" but potentially active configuration. This was confirmed by run-off transcription assays showing a restoration of c-fos and c-myc RNA synthesis when actinomycin D was displaced by proflavine.

Amanitins↗

PubMed1990

Affinity chromatography of mammalian and yeast nucleosomes. Two modes of binding of transcriptionally active mammalian nucleosomes to organomercurial-agarose columns, and contrasting behavior of the active nucleosomes of yeast.

The reasons for the selective binding of nucleosomes from transcriptionally active genes to the organomercurial-agarose columns have been investigated. At least two modes of binding are identified by a new two-stage elution procedure that discriminates between nucleosomes which are retained by the Hg-column because of their salt-labile associations with SH-reactive non-histone proteins, and nucleosomes in which a conformational change has made the thiol groups of histone H3 accessible to SH-reagents. The first class is released from the column in 0.5 M NaCl; the second class is eluted in 10 mM dithiothreitol which displaces the bound H3-thiols. In mammalian cells, both classes of Hg-bound nucleosomes are enriched in the DNA sequences being transcribed at the time, and their histones H3 and H4 are hyperacetylated. In yeast cells, in which histone H3 lacks cysteinyl residues, only a small fraction of nucleosomes binds to the mercury column, and it has no enrichment of DNA sequences derived from the actively transcribed GAL, HIS4, and ACT1 genes. Since few nucleosomes remain on the column after elution in 0.5 M NaCl, the bound nucleosomes of yeast are retained primarily because of salt-labile associations with thiol-reactive nonhistone proteins. Thus, the presence of histone H3-thiol groups appears to be essential for the mercury binding of the second class of nucleosomes which, in mammalian cells, is derived from the transcriptionally active genes. The results support models of reversible nucleosome unfolding during transcription in mammalian cells to reveal previously inaccessible H3-SH groups, and they also indicate that other thiol-containing proteins, including high mobility group 1 and 2, become closely but transiently associated with the chromatin subunits during their transcription.

Acetylation↗

PubMed1990

Nested allostery of arthropodan hemocyanin (Eurypelma californicum and Homarus americanus). The role of protons.

Continuous oxygen binding curves for two arthropodan hemocyanins were performed at different pH values ranging from 7.0 to 8.7 and in the presence of physiological concentrations of the bivalent ions Ca2+ and Mg2+. The arthropods Eurypelma californicum and Homarus americanus are classified as chelicerata and crustaceans, respectively. Their structurally well-characterized hemocyanins are composed of, in the case of E. californicum 24 subunits, and in the case of H. americanus 12 subunits. The role of protons as allosteric effectors of the oxygen binding was analysed in terms of the nesting model, which assumes hierarchies of allosteric equilibria that are based on obvious structural hierarchies. For each hemocyanin, the smallest structural repeating unit, the 12-mer or the 6-mer, respectively, was regarded as the "allosteric unit". Two allosteric units are allosterically coupled within the native molecules. The analysis revealed that in accordance with the postulations of the classical Monod-Wyman-Changeux model protons as allosteric effectors do not change the oxygen affinities of the four postulated conformations, but influence the allosteric equilibria between them at two different hierarchical levels. Model-independent determination of the affinity constants for the binding of the first and the last oxygen molecule to the native hemocyanins and to the isolated half-molecules confirmed the affinities calculated according to the nesting model. The stepwise establishment of new conformations during the assembly process from monomers to the structurally identical repeating unit and further on to the native molecule is shown. Possible physiological advantages of allosterically coupled allosteric units in contrast to allosterically uncoupled ones are thought to be (1) the option to regulate oxygen binding on different levels of structural hierarchy and (2) the increase of the oxygen-carrying capacity.

Allosteric Site↗

PubMed1990

[Hierarchies in the structure and function of oxygen-binding proteins].

The structures of respiratory proteins such as hemoglobins and hemocyanins show obvious hierarchies. They belong to the class of allosteric macromolecules with cooperative functional properties, as do key metabolic enzymes. When examining the molecular mechanisms on which allostery and cooperativity are based, it could be shown for arthropod hemocyanins that the structural hierarchy of these macromolecules is reflected in a functional hierarchy. This relationship is described quantitatively in the "nesting" model. This model also offers explanations for the physiological significance of the prominent hierarchy of these molecules.

Animals↗

PubMed1989

Estradiol enhanced acetylation of nuclear high mobility group proteins of the uterus of newborn guinea pigs.

The effect of estradiol on the acetylation of nuclear high mobility group (HMG) proteins in the uterus of newborn (3 days old) guinea pigs was studied "in vivo" and in tissue slices. In the "in vivo" studies after subcutaneous injection of 5 mCi [3H]-acetate there is a rapid (20 min) uptake of radioactive acetate in the HMG-1, HMG-2, HMG-14 and HMG-17 high mobility group proteins. In parallel studies, after administration of the same quantity of [3H]-acetate plus 20 micrograms of estradiol (E2), a selective increase in the acetylation of HMG-14 protein is observed. The preferential acetylation of HMG-14 can also be demonstrated in uterine tissue slices 20 minutes after exposure to the hormone (5 x 10(-8)M). In conclusion, the present data suggest that the acetylation of HMG proteins, in particular HMG-14, and like that of nucleosomal "core" histones, is an early event in gene activation by estradiol.

Acetates↗

PubMed1987

Affinity chromatographic purification of nucleosomes containing transcriptionally active DNA sequences.

The unfolding of nucleosome cores in transcriptionally active chromatin uncovers the sulfhydryl groups of histone H3, making them accessible to SH-reagents. This has suggested that nucleosomes from active genes could be retained selectively on organomercurial/agarose columns. When nucleosomes released from rat liver nuclei by limited digestion with micrococcal nuclease were passed through an Hg affinity column, a run-off fraction of compact, beaded nucleosomes was separated from a retained nucleosome fraction. Although both contained monomer-length DNA and a full complement of core histones, histones in the retained fraction were hyperacetylated. Dot blot hybridizations showed the Hg-bound nucleosome fraction to be enriched in DNA sequences transcribed by hepatocytes (serum albumin and transferrin genes), while a brain-specific gene (preproenkephalin) was not retained, but appeared in the nucleosomes of the run-off fraction. The results are discussed in light of other evidence linking hyperacetylation of histones H3 and H4 to conformational changes at the middle of the nucleosome core.

Acetylation↗

PubMed1987

Cell cycle-dependent changes in conformation and composition of nucleosomes containing human histone gene sequences.

Unfolding of the nucleosomes in transcriptionally active chromatin uncovers the sulfhydryl groups of histone H3 and permits the selective recovery of the unfolded nucleosomes by mercury-affinity chromatography. This new technique has been used to compare the nucleosomal proteins and their postsynthetic modifications in the unfolded and the compactly beaded nucleosomes of HeLa cells in logarithmic growth, and at different stages of the growth cycle. The Hg-bound nucleosomes are shown to be deficient in replicating DNA sequences, but to remain associated with fragments of nascent RNA chains (or RNP particles) during gradient centrifugations. Both nucleosome fractions contain a full complement of "core" histones but differ with respect to postsynthetic modifications. The Hg-bound nucleosomes contain high levels of the tri- and tetra-acetylated forms of histones H3 and H4. The unbound nucleosomes are deficient in acetylated histones but enriched in phosphorylated H2A. In synchronized HeLa cells, histone H2A and H4 gene sequences occur in the Hg-bound nucleosomes during the S-phase when their transcription takes place, but not in the G2-phase when the genes are repressed.

Acetylation↗

PubMed1987

Altered nucleosomes of active nucleolar chromatin contain accessible histone H3 in its hyperacetylated forms.

Chromatin of the organism Physarum polycephalum contains a class of conformationally altered nucleosomes previously localized to the transcribing regions of ribosomal genes in nucleoli. When nuclei are treated with 2-iodo[2-tritium]acetate, the histone H3 sulfhydryl group of the altered nucleosomes is derivatized while that of folded nucleosomes is not, and the labeled histones can then be identified by autoradiography of gels that separate H3 isoforms. The H3 derivatized is predominantly of tri- and tetraacetylated forms. In contrast, total free histone reacted with iodoacetate shows no preferential labeling of isoforms. Selective reaction of acetylated H3 is prevalent in both nucleolar and non-nucleolar chromatin. The results link specific patterns of H3 acetylation to changes in nucleosome conformation that occur during transcription.

Acetylation↗

PubMed1985

Methods for monitoring canine prostate size: internal and external caliper measurements.

A method has been developed that allows the generation of a three-dimensional representation of the canine prostate from a pair of orthogonal radiographs. This information can be used to externally measure the length, width, and height of the prostate and from these results to calculate prostate weight. This externally estimated prostate weight is correlated closely (r = 0.90) with the actual prostate weight. The ability to generate an external caliper measurement of prostate weight will facilitate future studies of the effect of aging and hormonal manipulation on the canine prostate.

Aging↗