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Mathias Sprinzl

Publications and source records attributed to Mathias Sprinzl.

16 recordsLinked to original sources
PubMed2006

Esterase 2-oligodeoxynucleotide conjugates as sensitive reporter for electrochemical detection of nucleic acid hybridization.

A thermostable, single polypeptide chain enzyme, esterase 2 from Alicyclobacillus acidocaldarius, was covalently conjugated in a site specific manner with an oligodeoxynucleotide. The conjugate served as a reporter enzyme for electrochemical detection of DNA hybridization. Capture oligodeoxynucleotides were assembled on gold electrode via thiol-gold interaction. The esterase 2-oligodeoxynucleotide conjugates were brought to electrode surface by DNA hybridization. The p-aminophenol formed by esterase 2 catalyzed hydrolysis of p-aminophenylbutyrate was amperometrically determined. Esterase 2 reporters allows to detect approximately 1.5 x 10(-18)mol oligodeoxynucleotides/0.6 mm2 electrode, or 3 pM oligodeoxynucleotide in a volume of 0.5 microL. Chemically targeted, single site covalent attachment of esterase 2 to an oligodeoxynucleotide significantly increases the selectivity of the mismatch detection as compared to widely used, rather unspecific, streptavidin/biotin conjugated proteins. Artificial single nucleotide mismatches in a 510-nucleotide ssDNA could be reliably determined using esterase 2-oligodeoxynucleotide conjugates as a reporter.

Base Pair Mismatch↗
PubMed2006

Interaction of immobilized human exportin-t with calf liver tRNA.

The binding of tRNA species from calf liver to immobilized exportin-t in the presence of ran x GppNHp was examined by affinity chromatography. We observed different eluting behaviors of individual tRNAs. After separating tRNAs on a two-dimensional polyacrylamide gel, the positions of seven selected tRNAs were identified by Northern hybridization and their relative affinities to immobilized exportin-t x ran x GppNHp complex were estimated in the order of tRNA(Leu)(CAG) > tRNA(Ser)(GCU), tRNA(Leu)(CAA), tRNA(Ser)(UGA) > tRNA(Ser)(AGA), tRNA(Leu)(AAG) > tRNA(Arg)(ACG). We propose that exportin-t preferentially binds and exports those tRNAs that are rapidly consumed by the protein synthesis machinery.

Animals↗
PubMed2006

Irreversible thermal denaturation of elongation factor Ts from Thermus thermophilus effect of the residual structure and intermonomer disulfide bond.

The homodimeric wild-type elongation factor Ts, EF-Ts(wt), and its C190A mutant, EF-Ts(C190A), from Thermus thermophilus goes through thermal denaturation in a way consistent with a two state irreversible model with a relatively high activation energy, approximately 530 kJ/mol (Supplemental materials provides a list of 98 activation energies from 54 proteins in various solvent conditions). Removing the intermonomeric disulfide bond by substituting alanine for cysteine 190 affects the rate constant of the irreversible thermal transition. At physiological temperatures, the half-life of the native conformations was estimated to be approximately 21 days for wt and 1.3 days for C190A. Thermally denatured EF-Ts refolds into a molten-globule-like state as indicated by its native-like circular dichroism spectrum in the far UV region and the enhanced fluorescence of the hydrophobic probe, 1-anilinonaphtalene-8-sulphonate. The residual secondary structure observed in the thermally denatured state of EF-Ts at high temperatures affects its apparent temperature of thermal transition, T(trs), independent of the presence or absence of the intermonomeric disulfide bond. The effect of the GdmHCl concentration on the activation energy, E(a), and the temperature, T*, i.e., the temperature at which the rate of the irreversible step is 1 min(-1), indicates that the intermonomeric disulfide bond contributes to the irreversibility of thermal transition of EF-Ts.

Algorithms↗
PubMed2006

C-terminal modifications of a protein by UAG-encoded incorporation of puromycin during in vitro protein synthesis in the absence of release factor 1.

Deactivation of release factor 1 by polyclonal antibodies in an in vitro translation system, which was used to express the esterase gene, led to the reversible elimination of naturally occurring termination. This technique allowed the antibiotic puromycin to be used as an acceptor substrate for the peptidyl residue in the peptidyl-transferase reaction. This resulted in more than 80 % yield of protein with C-terminally incorporated puromycin. pCpPuromycin that was either conjugated with the Cy3 fluorophor or biotin by N4 alkylation of cytosine, also acted as an acceptor substrate for the peptidyl-transferase reaction and was incorporated into the protein C terminus. The resulting conjugates possessed Cy3-specific fluorescence and affinity to streptavidin-coated surfaces, respectively. This left the enzymatic activity of the reporter protein unaffected. It was also shown that extension of puromycin on its 5'-hydroxyl end by up to ten deoxyoligonucleotides also allowed conjugation with the C terminus of in vitro translated protein when RF1-dependent termination was suppressed. However, the conjugation yield decreased upon addition of more than six nucleotides.

Animals↗
PubMed2005

Unusual effect of salts on the homodimeric structure of NADH oxidase from Thermus thermophilus in acidic pH.

The unusual salt-dependent behavior of the homodimeric flavoenzyme NADH oxidase from Thermus thermophilus in acidic pH has been studied using circular dichroism (CD) and sedimentation velocity. The native-like secondary and quaternary structures in acidic low ionic strength conditions were significantly perturbed by the addition of salts. The peptide region of the CD spectra showed a major salt-induced conformational change in the protein secondary structure. Sedimentation velocity experiments showed dissociation of the homodimeric structure of NADH oxidase in the presence of salt (>1 M). The new acidic conformation of the protein was stabilized by high ionic strength as indicated by a salt-induced increase in the melting temperature of the protein, and by a shift in the apparent pK(a) values of the conformational transition to a less acidic pH. Distortion of the dominant alpha-helical signal was expressed as the disappearance of the parallel polarized Moffitt exciton band at 208 nm without an accompanying loss of amplitude of n-->pi* electronic transitions at 222 nm. The unusual CD spectra correlated qualitatively with the theoretically calculated CD spectra of short alpha-helical structures and/or twisted beta-sheets. Differences between the experimentally obtained CD spectra and theoretical calculations (AGADIR) of the alpha-helical content of NADH oxidase indicate a role for non-local interactions in the protein conformation at high ionic strength and low pH. These findings indicate the importance of the homodimeric interface and electrostatic interactions for maintaining the structural integrity of this thermophilic protein.

Dimerization↗
PubMed2005

The esterase from Alicyclobacillus acidocaldarius as a reporter enzyme and affinity tag for protein biosynthesis.

Esterase from thermophilic bacteria Alicyclobacillus acidocaldarius can be produced up to 200 microg/ml by coupled in vitro transcription/translation system derived from Escherichia coli. The synthesized thermostable enzyme can be determined by photometrical and fluorescent assays at least up to 10(-8) M concentration or by activity staining in the polyacrylamide gels. Enhanced green fluorescence protein-esterase fusion protein was bound to a matrix with immobilized esterase inhibitor and purified by affinity chromatography. Thus, the esterase is suited as a reporter enzyme to monitor the expression of polypeptides coupled to its N-terminus and simultaneously, as a cleavable tag for polypeptide purification.

Affinity Labels↗
PubMed2005

Efficient suppression of the amber codon in E. coli in vitro translation system.

An mRNA encoding the esterase from Alicyclobacillus acidocaldarius with catalytically essential serine codon (ACG) replaced by an amber (UAG) codon was used to study the suppression in in vitro translation system. Suppression of UAG by tRNA(Ser(CUA)) was monitored by determination of the full-length and active esterase. It was shown that commonly used increase of suppressor tRNA concentration inhibits protein production and therefore limits suppression. In situ deactivation of release factor by specific antibodies leads to efficient suppression already at low suppressor tRNA concentration and allows an in vitro synthesis of fully active enzyme in high yield undistinguishable from wild-type protein.

Base Sequence↗
PubMed2005

Compilation of tRNA sequences and sequences of tRNA genes.

Maintained at the Universitat Bayreuth, Bayreuth, Germany, the Compilation of tRNA Sequences and Sequences of tRNA Genes is accessible at the URL http://www.tRNA.uni-bayreuth.de with mirror site located at the Institute of Protein Research, Pushchino, Russia (http://alpha.protres.ru/trnadbase). The compilation is a searchable, periodically updated database of currently available tRNA sequences. The present version of the database contains a new Genomic tRNA Compilation including the sequences of tRNA genes from genomic sequences published up to July 2003. It consists of about 5800 tRNA gene sequences from 111 organisms covering archaea, bacteria, higher and lower eukarya. The former Compilation of tRNA Genes (up to the end of 1998) and the updated Compilation tRNA Sequences (561 entries) are also supported by the new software. The database can be explored by using multiple search criteria and sequence templates. The database provides a service that allows to obtain statistical information on the occurrences of certain bases at given positions of the tRNA sequences. This allows phylogenic studies and search for identity elements in respect to interactions of tRNAs with various enzymes.

Animals↗
PubMed2004

Modulation of activity of NADH oxidase from Thermus thermophilus through change in flexibility in the enzyme active site induced by Hofmeister series anions.

The conformational dynamics of NADH oxidase from Thermus thermophilus was modulated by the Hofmeister series of anions (H2PO4-, SO42-, CH3COO-, Cl-, Br-, I-, ClO4-, SCN-) in the concentration range 0-3 M. Both chaotropic and kosmotropic anions, at high concentration, inhibit the enzyme by different mechanisms. Chaotropic anions increase the apparent Michaelis constant and decrease the activation barrier of the reaction. Kosmotropic anions have the opposite effect. Anions from the middle of the Hofmeister series do not significantly affect the enzyme activity even at high concentration. We detected no significant changes in ellipticity of the aromatic region in the presence of the anions studied. There is a decreased Stern-Volmer quenching constant for FAD fluorescence quenching in the presence of kosmotropic anions and an increased quenching constant in the presence of chaotropic anions. All of this indicates that active site flexibility is important in the function of the enzyme. The data demonstrate that both the high rigidity of the active site in the presence of kosmotropic anions, and its high flexibility in the presence of chaotropic anions have a decelerating effect on enzyme activity. The Hofmeister series of anions proved to be suitable agents for altering enzyme activity through changes in flexibility of the polypeptide chain, with potential importance in modulating extremozyme activity at room temperature.

Anions↗
PubMed2004

The tRNA world.

Explore the source record for details and available documents.

Animals↗
PubMed2003

Role of conformational flexibility for enzymatic activity in NADH oxidase from Thermus thermophilus.

NADH oxidase from Thermus thermophilus is a homodimer with an unknown physiological function. As is typical for an enzyme isolated from a thermophile, the catalytic rate, kcat, is low at low temperatures and increases with temperature, achieving an optimum at the physiological temperature of the organism, i.e. at approximately 70 degrees C for T. thermophilus. At low temperatures, the kcat of several enzymes from thermophilic and mesophilic organisms can be increased by chaotropic agents. The catalytic rate of NADH oxidase increases in the presence of urea. At concentrations of 1.0-1.3 m urea it reaches 250% of the activity in the absence of urea, at 20 degrees C. At higher urea concentrations the enzyme activity is inhibited. The urea-dependent activity changes correlate with changes in the fluorescence intensity of Trp47, which is located in the active site of the enzyme. Both fluorescence and circular dichroism measurements indicate that the activation by chaotropic agents involves local environmental changes accompanied by increased dynamics in the active site of the enzyme. This is not related to the global structure of NADH oxidase. The presence of an aromatic amino acid interacting with the flavin cofactor is common to numerous flavin-dependent oxidases. A comparison of the crystal structure with the activation thermodynamic parameters, deltaH* and TdeltaS*, obtained from the temperature dependence of kcat, suggests that Trp47 interacts with a water molecule and the isoalloxazine flavin ring. The present investigation suggests a model that explains the role of the homodimeric structure of NADH oxidase.

Binding Sites↗
PubMed2002

Thermodynamic properties of nucleotide-free EF-Tu from Thermus thermophilus in the presence of low-molecular weight effectors of its GTPase activity.

The thermal transition of elongation factor EF-Tu from Thermus thermophilus in the presence of low-molecular weight effectors was studied by differential scanning calorimetry. The effectors of GTPase activity used were the antibiotic kirromycin and the cations Li(+), Na(+), K(+) and NH(4)(+) in the chloride form. The temperature of thermal denaturation and the cooperativity of the transition of nucleotide-free EF-Tu (EF-Tu(f)) in the presence of kirromycin are comparable with those of the EF-Tu x guanosine-5'-[beta,gamma-imido]triphosphate (GppNHp) form, indicating similar conformational states. Increased concentrations of Na(+) and K(+) stabilized EF-Tu(f) in a manner similar to GppNHp. NH(4)(+) decreased the transition temperature of EF-Tu(f) and Li(+) decreased both the temperature and the calorimetric enthalpy of the thermal transition of EF-Tu(f). In the presence of salts, binding of kirromycin had a stabilizing effect on EF-Tu(f). Correlation between the GTPase activity and thermodynamic characteristics of EF-Tu(f) induced by kirromycin in the absence or presence of the cations is discussed.

Ammonium Chloride↗
PubMed2002

Microcalorimetric study of elongation factor Tu from Thermus thermophilus in nucleotide-free, GDP and GTP forms and in the presence of elongation factor Ts.

Elongation factor (EF) Tu undergoes profound nucleotide-dependent conformational changes in its functional cycle. The thermodynamic parameters of the different Thermus thermophilus EF-Tu forms, its domains I, II/III and III, were determined by microcalorimetry. Thermal transitions of the EF-Tu.GDP and EF-Tu.guanosine-5'-[beta,gamma-imido]triphosphate have a cooperative two-state character. Nucleotide removal affected the cooperativity of the thermal transition of EF-Tu. Microcalorimetric measurements of nucleotide-free EF-Tu and its separated domains showed that domains II/III have the main stabilizing role for the whole protein. Despite the fact that strong interactions between elongation factors Tu and Ts from T. thermophilus at 20 degrees C exist, the thermal transition of neither protein in the complex was significantly affected.

Calorimetry, Differential Scanning↗
PubMed2002

RNA aptamers directed against release factor 1 from Thermus thermophilus.

An in vitro selection/amplification (SELEX) was used to generate RNA aptamers that specifically bind Thermus thermophilus release factor 1 (RF1). From 31 isolated clones, two groups of aptamers with invariable sequences 5'-ACCU-3' and 5'-GAAAGC-3' were isolated. Chemical and enzymatic probing of the structure indicate that in both groups the invariable sequences are located in single-stranded regions of hairpin structures. Complex formations between RF1 and aptamers of both groups were identified by electrophoretic shift assay and chemical footprinting. Deletion of the invariable sequences did not effect the secondary structure of the aptamers but abolished their binding to RF1. RNA motifs matching the invariable sequences of the aptamers are present as consensus sequences in the peptidyl transferase center of 23S rRNAs. T. thermophilus RF1 recognizes UAG stop codons in an Escherichia coli in vitro translation system. Aptamers from both groups inhibited this RF1 activity.

Escherichia coli↗