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M Sprinzl

Publications and source records attributed to M Sprinzl.

At least 73 records · Page 4Linked to original sources
PubMed1993

Crystallization and preliminary X-ray diffraction studies of a NADH oxidase from Thermus thermophilus HB8.

The thermophile NADH oxidase from Thermus thermophilus, cloned and expressed in Escherichia coli, has been purified to homogeneity and crystallized. Three different crystal forms were found to be suitable for X-ray diffraction analysis. Crystals of the tetragonal form, grown in the presence of 25% polyethylene glycol 4000 and 0.25 M-NaCl at pH 6.6, were chosen for further analysis. These crystals belong to the space group P4(1)(3)2(1)2 with refined lattice constants of a = 94.8 A and c = 49.0 A, indicating a cell content of one monomer per asymmetric unit of the crystal. The crystals diffract to a resolution of 2.2 A.

Cloning, Molecular↗
PubMed1993

Participation of the overproduced elongation factor Tu from Thermus thermophilus in protein biosynthesis of Escherichia coli.

The influence of the overproduced elongation factor Tu (EF-Tu) from Thermus thermophilus on the protein biosynthesis in Escherichia coli was investigated both in vivo and in vitro. A kirromycin-resistant E. coli strain became sensitive to this antibiotic upon the expression of the tuf A-gene of T. thermophilus present on a plasmid. In in vitro translation with components of the kirromycin-resistant E. coli strain the poly(Phe) synthesis stopped when minute amounts of the EF-Tu from T. thermophilus were added. Both results indicate the sensitivity of the T. thermophilus EF-Tu to kirromycin and its participation in the polypeptide synthesis of E. coli.

Bacterial Proteins↗
PubMed1993

Selection for active E. coli tRNA(Phe) variants from a randomized library using two proteins.

In vitro selection was used to isolate active Escherichia coli tRNA(Phe) variants from randomized libraries. Functional tRNAs were first selected by multiple rounds of binding to Escherichia coli phenylalanyl-tRNA synthetase. These variants were then aminoacylated and selected for affinity to elongation factor-Tu. By randomizing potential recognition nucleotides, the importance of residues U20, G34, A35, A36 and U59, previously identified to be required for specific recognition by E. coli phenylalanyl-tRNA synthetase (FRS), was confirmed. However, the sequences of several active variants imply that the wild-type tertiary interactions G10-C25-U45 and A26-G44 are not required for recognition, as previously suggested. Selection of functional tRNAs from a second library randomized at positions normally involved in conserved tertiary interactions revealed new combinations of nucleotides at these positions, suggesting the presence of novel tertiary interactions. In both libraries, active sequences containing deletions were isolated. Taken together, it is clear that FRS is active with substrates having an unexpectedly broad sequence diversity. Finally, the potency of this method is illustrated by the identification of a second class of variants that was isolated by virtue of the presence of an impurity in the FRS preparation.

Base Sequence↗
PubMed1993

Ternary complex between elongation factor Tu.GTP and Phe-tRNA(Phe).

The effect of aminoacylation and ternary complex formation with elongation factor Tu.GTP on the tertiary structure of yeast tRNA(Phe) was examined by 1H-NMR spectroscopy. Esterification of phenylalanine to tRNA(Phe) does not lead to changes with respect to the secondary and tertiary base pair interactions of tRNA. Complex formation of Phe-tRNA(Phe) with elongation factor Tu.GTP results in a broadening of all imino proton resonances of the tRNA. The chemical shifts of several NH proton resonances are slightly changed as compared to free tRNA, indicating a minor conformational rearrangement of Phe-tRNA(Phe) upon binding to elongation factor Tu.GTP. All NH proton resonances corresponding to the secondary and tertiary base pairs of tRNA, except those arising from the first three base pairs in the aminoacyl stem, are detectable in the Phe-tRNA(Phe)-elongation factor Tu-GTP ternary complex. Thus, although the interactions between elongation factor Tu and tRNA accelerate the rate of NH proton exchange in the aminoacyl stem-region, the Phe-tRNA(Phe) preserves its typical L-shaped tertiary structure in the complex. At high (> 10(-4) M) ligand concentrations a complex between tRNA(Phe) and elongation factor Tu-GDP can be detected on the NMR time-scale. Formation of this complex is inhibited by the presence of any RNA not related to the tRNA structure. Using the known tertiary structures of yeast tRNA(Phe) and Thermus thermophilus elongation factor Tu in its active, GTP form, a model of the ternary complex was constructed.

Acylation↗
PubMed1992

Structure of the phenylalanyl-tRNA synthetase genes from Thermus thermophilus HB8 and their expression in Escherichia coli.

A 4459 bp long BamHI restriction fragment containing the two genes for the Thermus thermophilus HB8 phenylalanyl-tRNA synthetase was cloned in Escherichia coli and its nucleotide sequence was determined. The genes pheS and pheT encode the alpha- and beta-subunits with a molecular weight of 39 and 87 kD, respectively. Three conserved sequence motifs typical for class II tRNA synthetases occur in the alpha-subunit. Secondary structure predictions indicate that an arm composed of two anti-parallel alpha-helices similar to that reported for the E.coli seryl-tRNA synthetase may be present in its N-terminal portion. In the beta-subunit clusters of hydrophilic amino acids and a leucine zipper motif were identified, and several pronounced alpha-helical regions were predicted. The particular arginine and lysine residues in the N-terminal portion of the beta-subunit, which were found to participate in tRNA binding in the yeast and E.coli PheRSs, have their counterparts in the T.thermophilus protein. The 5'-portion of an open reading frame downstream of pheT was found and codes for a yet unidentified, extremely hydrophobic peptide. The pheST genes are presumably cotranscribed and translationally coupled. A novel type of a putative transcriptional terminator in Thermus species was identified immediately downstream of pheT and other Thermus genes. The genes pheS and pheST were expressed in E.coli.

Amino Acid Sequence↗
PubMed1992

Affinity labeling of GTP-binding proteins in cellular extracts.

GTP-binding proteins in cellular extracts from Escherichia coli, Thermus thermophilus, yeast, wheat germ or calf thymus were identified using in situ periodate-oxidized [alpha-32P]GTP as affinity label. Site-specific reaction of individual GTP-binding proteins was achieved by cross-linking the protein-bound 2',3'-dialdehyde derivative of GTP with the single lysine residue of the conserved NKXD sequence through Schiff's base formation and subsequent cyanoborohydride reduction. Labeled GTP-binding proteins from prokaryotic or eukaryotic cell homogenates were separated by polyacrylamide gel electrophoresis and visualized by autoradiography. In addition cross-linking of [alpha-32P]GTP with GTP-binding proteins was demonstrated in model systems using different purified GTPases, human c-H-ras p21, transducin from bovine retina, polypeptide elongation factor Tu (EF-Tu) from T. thermophilus and initiation factor 2 (IF2) from T. thermophilus. The described affinity labeling technique can serve as an analytical method for the identification of GTPases belonging to the classes of ras-proteins, elongation and initiation factors, and heterotrimeric signal transducing G-proteins.

Affinity Labels↗
PubMed1992

Molecular cloning and nucleotide sequence of the gene encoding a H2O2-forming NADH oxidase from the extreme thermophilic Thermus thermophilus HB8 and its expression in Escherichia coli.

The sequence of the 32 N-terminal amino acids of the NADH oxidase from the extreme thermophile, Thermus thermophilus HB8, was used to synthesize oligonucleotides to probe for the respective gene in a genomic library of T. thermophilus HB8. The gene encoding the NADH oxidase, designated nox, was cloned, its nucleotide sequence was determined and found to be colinear with the N-terminal sequence of the enzyme. The molecular mass of 26835 Da, as deduced from the nox gene, agrees with that of the purified NADH oxidase from T. thermophilus HB8 (25,000 Da), as estimated by polyacrylamide gel electrophoresis under denaturing conditions. The nox gene was overexpressed in Escherichia coli and a protocol for the rapid purification of the enzyme was developed. The E. coli-borne T. thermophilus HB8 NADH oxidase has properties identical to those of the authentic T. thermophilus HB8 enzyme and possesses a high thermal stability.

Amino Acid Sequence↗
PubMed1992

Purification and characterization of a NADH oxidase from the thermophile Thermus thermophilus HB8.

A NADH oxidase has been purified from the extreme thermophile Thermus thermophilus HB8 by several chromatographic steps. The purified enzyme was essentially homogeneous as judged by gel electrophoresis under denaturing conditions and by determination of the N-terminal amino acids sequence. It is a monomeric flavin-adenine-dinucleotide-containing flavoprotein with an apparent molecular mass of 25 kDa and an 1:1 ratio of FAD to the polypeptide chain. The purified enzyme catalyzes the oxidation of reduced NADH or NADPH with the formation of H2O2. The apparent Km values for NADH and NADPH are 4.14 microM and 14.0 microM (pH 7.2 at room temperature), respectively, with a sixfold greater kcat/Km values for NADH compared to NADPH. The enzyme uses O2 as an electron acceptor in the presence of either FAD, riboflavin 5'-phosphate or riboflavin as cofactor. In addition, the enzyme is able to catalyze electron transfer from NADH to various other electron acceptors (methylene blue, cytochrome c, p-nitroblue tetrazolium, 2,6-dichloroindophenol and potassium ferricyanide), even in the absence of flavin shuttles. No significant inhibition of the NADH oxidoreductase activity by superoxide dismutase was observed with these artificial electron acceptors, indicating that electron transfer occurs mainly from NADH directly to the electron acceptors, not via O2- as an intermediate. The purified NADH oxidase exhibits highest activity at pH 5.0 and is stable at elevated temperatures of up to 80 degrees C.

Amino Acid Sequence↗
PubMed1992

Nucleotide binding and GTP hydrolysis by elongation factor Tu from Thermus thermophilus as monitored by proton NMR.

Proton NMR experiments of the GTP/GDP-binding protein EF-Tu from the extremely thermophilic bacterium Thermus thermophilus HB8 in H2O have been performed paying special attention to the resonances in the downfield region (below 10 ppm). Most of these downfield signals are due to hydrogen bonds formed between the protein and the bound nucleotide. However, three downfield resonances appear even in the nucleotide-free EF-Tu. The middle and C-terminal domain (domain II/III) of EF-Tu lacking the GTP/GDP-binding domain gives rise to an NMR spectrum that hints at a well-structured protein. In contrast to native EF-Tu, the domain II/III spectrum contains no resonances in the downfield region. Several downfield resonances can be used as a fingerprint to trace hydrolysis of protein-bound GTP and temperature effects on the EF-Tu.GDP spectra. NMR studies of the binding of guanosine nucleotide analogues (GMPPNP, GMPPCP) to nucleotide-free EF-Tu have been carried out. The downfield resonances of these complexes differ from the spectrum of EF-Tu.GTP. Protected and photolabile caged GTP was bound to EF-Tu, and NMR spectra before and after photolysis were recorded. The progress of the GTP hydrolysis could be monitored using this method. The downfield resonances have been tentatively assigned taking into account the known structural and biochemical aspects of EF-Tu nucleotide-binding site.

Amino Acid Sequence↗
PubMed1992

NMR study of the phosphate-binding loops of Thermus thermophilus elongation factor Tu.

The phosphoryl-binding loops in the guanosine diphosphate binding domain of elongation factor Tu were studied by 15N heteronuclear proton-observe NMR methods. Five proton resonances were found below 10.5 ppm. One of these was assigned to the amide group of Lys 24, which is a conserved residue in the phosphoryl-binding concensus loop of purine nucleotide binding proteins. The uncharacteristic downfield proton shift is attributed to a strong hydrogen bond with a phosphate oxygen. The amide protons from the homologous lysines in N-ras p21 [Redfield, A.G., & Papastavros, M.Z. (1990) Biochemistry 29, 3509-3514] and the catalytic domain of Escherichia coli elongation factor Tu [Lowry, D.F., Cool, R.H., Redfield, A.G., & Parmeggiani, A. (1991) Biochemistry 30, 10872-10877] also resonate downfield in similar positions. We propose that the downfield shift of this lysine amide proton is a spectral marker for this class of proteins. We also have studied the temperature dependence of the downfield resonances and find a possible conformation change at 40 degrees C.

Binding Sites↗
PubMed1991

Crystals of intact elongation factor Tu from Thermus thermophilus diffracting to high resolution.

The intact elongation factor Tu from the extreme thermophile Thermus thermophilus has been crystallized as a complex with the GTP analogue guanosine-5'-(beta,gamma-imido)triphosphate. The crystals are very stable in the X-ray beam and diffract to 1.9 A resolution. They exhibit space group C2, with a = 150.3(6) A, b = 99.6(3) A, c = 40.1(1) A, beta = 95.4(2) degrees, and contain one elongation factor Tu molecule per asymmetric unit.

Crystallization↗
PubMed1991

Effect of Thermus thermophilus elongation factor Ts on the conformation of elongation factor Tu.

Affinity labeling in situ of the Thermus thermophilus elongation factor Tu (EF-Tu) nucleotide binding site was achieved with periodate-oxidized GDP (GDPoxi) or GTP (GTPoxi) in the absence and presence of elongation factor Ts (EF-Ts). Lys52 and Lys137, both reacting with GDPoxi and GTPoxi, are located in the nucleotide binding region. In the absence of EF-Ts Lys137 and to a lesser extent Lys52 were accessible to the reaction with GTPoxi. GDPoxi reacted much more efficiently with Lys52 than with Lys137 under these conditions [Peter, M. E., Wittman-Liebold, B. & Sprinzl, M. (1988) Biochemistry 27, 9132-9138]. In the presence of EF-Ts, GDPoxi reacted more efficiently with Lys137 than with Lys52, indicating that the interaction of EF-Ts with EF-Tu.GDPoxi induces a conformation resembling that of the EF-Tu.GDPoxi complex in the absence of EF-Ts. Binding of EF-Ts to EF-Tu.GDP enhances the accessibility of the Arg59-Gly60 peptide bond of EF-Tu to trypsin cleavage. Hydrolysis of this peptide bond does not interfere with the ability of EF-Ts to bind to EF-Tu. EF-Ts is protected against trypsin cleavage by interaction with EF-Tu.GDP. High concentrations of EF-Ts did not interfere significantly with aminoacyl-tRNA.EF-Tu.GTP complex formation.

Affinity Labels↗
PubMed1991

An alkaline phosphatase protection assay to investigate trp repressor/operator interactions.

We have used an alkaline phosphatase protection assay to investigate the interaction of the trp repressor with its operator sequence. The assay is based on the principle that the trp repressor will protect a terminally 5'-32P-labeled operator DNA fragment from attack by alkaline phosphatase. The optimal oligonucleotide for investigating the trp repressor/operator interaction extends two base pairs from each end of the genetically defined target sequence predicted by in vivo studies [Bass et al. (1987) Genes Dev. 1, 565-572]. The assay works well over a 10,000-fold range of protein/DNA affinity and is used to show that the corepressor, L-tryptophan, causes the liganded repressor to bind a 20 base pair trp operator duplex 6400 times more strongly than the unliganded aporepressor. The affinity of the trp repressor for operators containing symmetrical mutations was interpreted in terms of the trp repressor/operator crystal structure as follows: (1) Direct hydrogen bonds with the functional groups of G-9 of the trp operator and the side chain of Arg 69 of the trp repressor contribute to DNA-binding specificity. (2) G-6 of the trp operator is critical for DNA-binding specificity probably because of the two water-mediated hydrogen bonds between its functional groups and the N-terminus of the trp repressor's E-helix. (3) Sequence-dependent aspects of the trp operator's conformation help stabilize the trp repressor/operator complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1991

Localization of a specific nucleotide in yeast tRNA by scanning transmission electron microscopy using an undecagold cluster.

Scanning transmission electron microscopic images of transfer RNAs reveal the molecular dimensions and compact morphology of these small macromolecules in unprecedented detail. Selective labeling of a sulfhydryl group on 2-thiocytidine enzymatically inserted at position 75 at the 3' end of yeast tRNA(Phe) with an undecagold cluster permits identification of this specific tRNA site by dark field STEM. Imaging of a single nucleotide at a defined location on the tRNA molecule should make it possible to localize in situ tRNAs at the A, P, and E sites of the ribosomal peptidyl transferase center, and in complexes of tRNA with enzymes and elongation factors. In addition, this approach may be used for the highly specific topographical mapping of other RNAs and/or biological macromolecular complexes.

Gold↗
PubMed1990

Interaction of the isolated domain II/III of Thermus thermophilus elongation factor Tu with the nucleotide exchange factor EF-Ts.

The middle and C-terminal domain (domain II/III) of elongation factor Tu from Thermus thermophilus lacking the GTP/GDP binding domain have been prepared by treating nucleotide-free protein with Staphylococcus aureus V8 protease. The isolated domain II/III of EF-Tu has a compact structure and high resistance against tryptic treatment and thermal denaturation. As demonstrated by circular dichroism spectroscopy, the isolated domain II/III does not contain any alpha-helical structure. Nucleotide exchange factor, EF-Ts, was found to interact with domain II/III, whereas the binding of aminoacyl-tRNA, GDP and GTP to this EF-Tu fragment could not be detected.

Circular Dichroism↗