Biomedical subjects

M Sprinzl

Publications and source records attributed to M Sprinzl.

At least 19 recordsLinked to original sources

PubMed2006

RNA aptamers directed against oligosaccharides.

Nucleic acid molecules are designed to interact predominantly with proteins or complementary nucleic acids. Interaction of nucleic acids with carbohydrates, abundant constituents of glycoproteins and glycolipids, are not common in cells. Biomedical applications of nucleic acids targeted against oligosaccharides, which are involved in the function of receptors, immune answer, host interaction with invading infectious agents, and cancer metastasis, are feasible. In vitro selection of nucleic acids interacting with oligoand polysaccharides is a promising strategy to identify potential inhibitors of biochemical recognition processes in which carbohydrates are involved. Several RNA and DNA aptamers directed against carbohydrates have already been isolated and characterized. The results are summarized in this article, and an attempt is made to draw initial conclusions concerning the perspectives of the outlined approach.

Animals↗

PubMed2005

pH-induced changes in activity and conformation of NADH oxidase from Thermus thermophilus.

Thermus thermophilus NADH oxidase (NOX) activity exhibits a bell-shaped pH-dependency with the maximal rate at pH 5.2 and marked inhibition at lower pH. The first pH transition, from pH 7.2 to pH 5.2, results in more than a 2-fold activity increase with protonation of a group with pKa=6.1+/-0.1. The difference in fluorescence of the free and enzyme-bound flavin strongly indicates that the increase in enzyme activity in a pH-dependent manner is related to a protein-cofactor interaction. Only one amino acid residue, His75, has an intrinsic pKa approximately 6.0 and is localized in proximity (<10 A) to N5-N10 of the isoalloxazine ring and, therefore, is able to participate in such an interaction. Solvent acidification leads to the second pH transition from pH 5.2 to 2.0 that results in complete inhibition of the enzyme with protonation of a group with an apparent pKa=4.0+/-0.1. Inactivation of NOX activity at low pH is not caused by large conformational changes in the quaternary structure as judged by intrinsic viscosity and sedimentation velocity experiments. NOX exists as a dimer even as an apoprotein at acidic conditions. There is a strong coupling between the fluorescence of the enzyme-bound flavin and the intrinsic tryptophans, as demonstrated by energy transfer between Trp47 and the isoalloxazine ring of flavin adenine dinucleotide (FAD). The pH-induced changes in intrinsic tryptophan and FAD fluorescence indicate that inhibition of the FAD-binding enzyme at low pH is related to dissociation of the flavin cofactor, due to protonation of its adenine moiety.

Adenine↗

PubMed2001

Effect of the central disulfide bond on the unfolding behavior of elongation factor Ts homodimer from Thermus thermophilus.

Functionally active elongation factor Ts (EF-Ts) from Thermus thermophilus forms a homodimer. The dimerization interface of EF-Ts is composed of two antiparallel beta-sheets that can be connected by an intermolecular disulfide bond. The stability of EF-Ts from T. thermophilus in the presence and absence of the intermolecular disulfide bond was studied by differential scanning calorimetry and circular dichroism. The ratio of the van't Hoff and calorimetric enthalpies, delta H(vH)/delta H(cal), indicates that EF-Ts undergoes thermal unfolding as a dimer independently of the presence or absence of the disulfide bond. This can be concluded from (1) the presence of residual secondary structure above the thermal transition temperature, (2) the absence of concentration dependence, which would be expected for dissociation of the dimer prior to unfolding of the monomers, and (3) a relatively low heat capacity change (delta Cp) upon unfolding. The retained dimeric structure of the thermally denatured state allowed for the determination of the effect of the intermolecular disulfide bond on the conformational stability of EF-Ts, which is deltadelta G(S-S,SH HS) = 10.5 kJ/mol per monomer at 72.5 degrees C. The possible physiological implications of the dimeric EF-Ts structure and of the intersubunit disulfide bond for the extreme conformational stability of proteins in thermophiles are discussed.

Bacterial Proteins↗

PubMed2001

Effect of N-domain on the stability of elongation factor Ts from Thermus thermophilus.

Elongation factor Ts (EF-Ts) from Thermus thermophilus forms a stable, functionally active homodimer in solution. Its monomer is composed of two domains: amino-terminal domain containing 50 amino acid residues and a larger, 146 residues long, C-domain which participates in dimerization of EF-Ts. Effect of removal of the N-domain on the conformational stability of EF-Ts has been studied. For comparison, the stabilities of both the full-length EF-Ts and its C-domain were studied by differential scanning calorimetry, electronic absorption and fluorescence spectroscopies over a pH range from 4 to approximately 13. Thermal denaturation of EF-Ts and of C-domain, followed by circular dichroism at 222 nm, at pH 7.0, and the pH dependence of the fluorescence of the single tryptophan 30 residue indicate a conformational instability of the N-domain. While N-domain does not affect the stability of full-length EF-Ts at acidic pH, its removal leads to stabilization of the rest of the protein at basic pH. This is reflected by higher values of transition temperatures and calorimetric enthalpies of C-domain as compared to the full-length EF-Ts. High mobility of the N-domain in alkaline pH conditions decreased the thermal stability of covalently linked C-domain of EF-Ts. An increase in intramolecular interactions at acidic pH together with a decrease of conformational entropies of the thermally denatured proteins most likely diminishes this destabilization effect.

Calorimetry, Differential Scanning↗

PubMed2001

Translation ability of mitochondrial tRNAsSer with unusual secondary structures in an in vitro translation system of bovine mitochondria.

BACKGROUND: Metazoan mitochondrial (mt) tRNAs are structurally quite different from the canonical cloverleaf secondary structure. The mammalian mt tRNASerGCU for AGY codons (Y = C or U) lacks the entire D arm, whereas tRNASerUGA for UCN codons (N = A, G, C or U) has an extended anti-codon stem. It has been a long-standing problem to prove experimentally how these tRNAsSer work in the mt translation system. RESULTS: To solve the above-mentioned problem, we examined their translational abilities in an in vitro bovine mitochondrial translation system using transcripts of altered tRNASer analogues derived from bovine mitochondria. Both tRNASer analogues had almost the same ability to form ternary complexes with mt EF-Tu and GTP. The D-arm-lacking tRNASer GCU analogue had considerably lower translational activity than the tRNASerUGA analogue and produced mostly short oligopeptides, up to a tetramer. In addition, tRNASerGCU analogue was disfavoured by the ribosome when other tRNAs capable of decoding the cognate codon were available. CONCLUSION: Both mt tRNASerGCU and tRNASerUGA analogues with unusual secondary structure were found to be capable of translation on the ribosome. However, the tRNASerGCU analogue has some molecular disadvantage on the ribosome, which probably derives from the lack of a D arm.

Animals↗

PubMed2001

Fluorescence-monitored conformational change on the 3'-end of tRNA upon aminoacylation.

Fluorescent tRNAs species with formycine in the 3'-terminal position (tRNA-CCF) were derived from Escherichia coli tRNA(Val). Thermus thermophilus tRNA(Aap) and Thermus thermophilus tRNA(Phe). The fluorescence of formycine was used to monitor the conformational changes at the 3'-terminus of tRNA caused by aminoacylation and hydrolysis of aminoacyl residue from aminoacyl-tRNAs. An increase of about 15% in the fluorescence intensity was observed after aminoacylation of the three tRNA-CCF. This change in fluorescence amplitude that is reversed by hydrolysis of the aminoacyl residue, does not depend on the structure of the amino acid or tRNA sequence. A local conformational change at the 3'-terminal formycine probably involving a partial destacking of the base moiety in the ACCF end takes place as a consequence of aminoacylation. A structural change at the 3'-terminus of tRNA induced by attachment and detachment of the acyl residue may be important in controlling the substrate/product relationship in reactions in which tRNA participates during protein biosynthesis.

Chromatography, High Pressure Liquid↗

PubMed2000

Interaction of fMet-tRNAfMet and fMet-AMP with the C-terminal domain of Thermus thermophilus translation initiation factor 2.

Two polypeptides resistant against proteolytic digestion were identified in Thermus thermophilus translation initiation factor 2 (IF2): the central part of the protein (domains II/III), and the C-terminal domain (domain IV). The interaction of intact IF2 and the isolated proteolytic fragments with fMet-tRNAfMet was subsequently characterized. The isolated C-terminal domain was as effective in binding of the 3' end of fMet-tRNAf Met as intact IF2. N-Formylation of Met-tRNAfMet was required for its efficient binding to the C-terminal domain. This suggests that the interaction between the C-terminal domain and the 3' end of fMet-tRNAfMet is responsible for the recognition of fMet-tRNAfMet by IF2 during translation initiation. Moreover, it was demonstrated that fMet-AMP is a minimal ligand of IF2. fMet-AMP inhibits fMet-tRNAfMet binding to IF2 as well as the activity of IF2 in the stimulation of ApUpG-dependent ribosomal binding of fMet-tRNAf Met. Specific interaction of fMet-AMP with IF2 was demonstrated by 1H-NMR spectroscopy. These findings indicate that fMet-AMP and the 3' terminal fMet-adenosine of fMet-tRNAfMet use the same binding site on the C-terminal domain of IF2 and imply that the interaction between the C-terminal domain and the 3' end of fMet-tRNAfMet is primarily responsible for the fMet-tRNAfMet binding and recognition by IF2.

Adenosine Monophosphate↗

PubMed1999

Two novel point mutations of mitochondrial tRNA genes in histologically confirmed Parkinson disease.

Mutations in mitochondrially encoded tRNA genes have been described in a variety of neurological disorders. One such mutation, the A to G transition at nucleotide position 4336 of the mitochondrial tRNA(Gln) gene, has been associated with both Alzheimer and Parkinson disease. We have now performed a complete sequence analysis of all 22 mitochondrially encoded tRNA genes in 20 cases of histologically proven idiopathic Parkinson disease. Genomic DNA extracted from the substantia nigra of frozen or formalin-fixed and paraffin-embedded brains was used for amplification by polymerase chain reaction followed by automated sequencing. Two new homoplasmic point mutations were detected in the genes for tRNA(Thr) (15950 G/A) and tRNA(Pro) (15965 T/C) in 1 patient each. Restriction enzyme digestion revealed absence of the 15950 G/A mutation in 96 controls and in 40 cases of neuropathologically confirmed Alzheimer disease. The 15965 T/C mutation was shown to be absent from 100 control subjects and 47 Alzheimer cases. In addition to the two novel mutations, six known sequence variants were detected in a total of 6 different patients in the genes for tRNA(Asp) (G7521A, 1), tRNA(Arg) (T10463C, 1), tRNA(LeuCUN) (A12308G, 2), and tRNA(Thr) (A15924G, 1; G15928A, 2), including 1 patient carrying the tRNA(Gln) (A4336G) mutation. The G15950A transition affects position 70 of the aminoacyl acceptor stem of tRNA(Thr), which has been implicated as a recognition element for threonyl-tRNA synthetase and, at least in some tRNAs, in the processing of primary mitochondrial transcripts. The T15965C point mutation in the mitochondrial tRNA(Pro) gene alters position 64 of the TpsiC stem. The corresponding nucleotide in bacterial aminoacyl-tRNAs is involved in the interaction with elongation factor Tu. Thus, the two novel mutations are likely to be of functional relevance and could contribute to dopaminergic nerve cell death in affected individuals.

Aged↗

PubMed1999

Crystal structure of acceptor stem of tRNA(Ala) from Escherichia coli shows unique G.U wobble base pair at 1.16 A resolution.

The acceptor stem of Escherichia coli tRNA(Ala), rGGGGCUA.rUAGCUCC (ALAwt), contains the main identity element for the correct aminoacylation by the alanyl tRNA synthetase. The presence of a G3.U70 wobble base pair is essential for the specificity of this reaction, but there is a debate whether direct minor-groove contact with the 2-amino group of G3 or a distortion of the acceptor stem induced by the wobble pair is the critical feature recognized by the synthetase. We here report the structure analysis of ALAwt at near-atomic resolution using twinned crystals. The crystal lattice is stabilized by a novel strontium binding motif between two cis-diolic O3'-terminal riboses. The two independent molecules in the asymmetric unit of the crystal show overall A-RNA geometry. A comparison with the crystal structure of the G3-C70 mutant of the acceptor stem (ALA(C70)) determined at 1.4 A exhibits a modulation in ALAwt of helical twist and slide due to the wobble base pair, but no recognizable distortion of the helix fragment distant from the wobble base pair. We suggest that a highly conserved hydration pattern in both grooves around the G3.U70 wobble base pair may be functionally significant.

Base Pairing↗

PubMed1999

Disorder and twin refinement of RNA heptamer double helices.

An RNA helix with seven base pairs which was derived from the acceptor stem of Escherichia coli tRNA(Ala), rGGGGCUA.rUAGCUCC (ALA(wt)), as well as a variant, rGGGGCUA.rUAGCCCC (ALA(C70)), in which the single G.U wobble base pair of ALA(wt) was replaced by G.C, crystallize in space group C2. Both non-isomorphic crystal forms display a complex packing pattern, which can be described alternatively as disorder or pseudo-merohedral twinning. The structure of ALA(wt) was determined by SIRAS phasing using an isomorphous iodine derivative, rGGGGCi(5)UA.rUAGCUCC (ALA(I)). All three RNA structures were subsequently subjected to twin refinement in space group P1, using anisotropic thermal displacement parameters at resolutions of 1.16, 1.23 and 1.4 A for ALA(wt), ALA(I) and ALA(C70), respectively. Alternatively, the structure of ALA(wt) was refined in space group C2 assuming twofold disorder of the molecular orientation. The refined structures are of reasonable quality according to all available indicators. There are no systematic differences between the molecular models resulting from twin refinement and disorder refinement.

Base Sequence↗

PubMed1998

Dimers of Thermus thermophilus elongation factor Ts are required for its function as a nucleotide exchange factor of elongation factor Tu.

Elongation factor Ts (EF-Ts) promotes the formation of active GTP-bound elongation factor Tu (EF-Tu) by accelerating the dissociation of GDP from the EF-Tu x GDP complex. Thermus thermophilus EF-Ts forms a dimer in solution, which is stabilised by interaction of a three-stranded antiparallel beta-sheet from each of the two EF-Ts molecules. A disulfide bridge and several hydrophobic interactions are the main structural elements which stabilise the dimer [Jiang, Y., Nock, S., Nesper, M., Sprinzl, M. & Sigler, P. B. (1996) Biochemistry 35, 10269-10278]. Site-directed mutagenesis was used to study the dimer formation and the effect of dimerization on the nucleotide exchange activity. The presence of the covalent disulfide bridge between the Cys190 residues has no effect on the activity. However, this disulfide bridge is not a necessary condition for the activity of EF-Ts. The amino acid residues Leu73, Cys190 and Phe192 form a hydrophobic core on the dimerization interface. Their replacement by Asp, Ala and Asp, respectively, influences to different degrees the stability of EF-Ts, the ability of EF-Ts to form dimers, and the ability to interact with EF-Tu. EF-Ts variants which were unable to form dimers were also inactive in the nucleotide exchange on EF-Tu. CD spectroscopy confirmed that this loss of activity is not due to changes in EF-Ts secondary structure. By calorimetric measurements, it was demonstrated that the dimer formation considerably contributes to the thermostability of T. thermophilus EF-Ts. Dimerization of T. thermophilus EF-Ts is required to fulfil its physiological function in protein biosynthesis and probably represents a strategy of the translation system in this thermophile to withstand high temperatures. The biochemical data presented here are supported by the recently solved structure of the T. thermophilus EF-Tu x EF-Ts complex [Wang, Y., Jiang, Y., Meyering-Voss, M., Sprinzl, M. & Sigler, P. B. (1997) Nature Struct. Biol. 4, 650-656] in which each EF-Tu in the dyad symmetrical heterotetrameric complex interacts with two subunits of EF-Ts.

Bacterial Proteins↗

PubMed1998

In vitro selected RNA molecules that bind to elongation factor Tu.

RNA molecules which bind to elongation factor Tu from T. thermophilus were isolated from a pool of ribooligonucleotides with a randomized sequence region. These RNAs interact with elongation factor Tu in both the GTP and the GDP form. A slight preference for the GTP form of the protein was observed. The isolated RNA aptamers compete with each other for a common binding site on elongation factor Tu. This binding site is different from the binding site for aminoacyl-tRNA or the binding site for elongation factor Ts and is located on domain II of elongation factor Tu. The selected RNAs do not bind to elongation factor G. The EF-Tu binding RNAs share a short consensus sequence, 5'-ACCGAAG-3', which was also found in the alpha-sarcin domain of T. thermophilus23S rRNA. The isolated RNAs have a hairpin structure with the 5'-ACCGAAG-3' sequence located in non-base-paired regions. Chemical probing and deletion experiments indicate that the consensus sequence is required for the interaction with elongation factor Tu.

Base Sequence↗

PubMed1998

Compilation of tRNA sequences and sequences of tRNA genes.

Sequences of 3279 sequences of tRNA genes and tRNAs published up to December 1996 are included in the compilation. Alignment of the sequences, which is most compatible with the tRNA phylogeny and known three-dimensional structures of tRNA, is used. Sequences and references are available under http://www.uni-bayreuth. de/departments/biochemie/trna/

Animals↗

PubMed1998

Initiation factors of protein biosynthesis in bacteria and their structural relationship to elongation and termination factors.

Initiation of protein biosynthesis in bacteria requires three initiation factors: initiation factor 1, initiation factor 2 and initiation factor 3. The mechanism by which initiation factors form the 70S initiation complex with initiator fMet-tRNA(fMet) interacting with the initiation codon in the ribosomal P site and the second mRNA codon exposed in the A site is not yet understood. Here, we present a model for the function of initiation factors 1 and 2 that is based on the analysis of sequence homologies, biochemical evidence and the present knowledge of the three-dimensional structures of translation factors and ribosomes. The model predicts that initiation factors 1 and 2 interact with the ribosomal A site mimicking the structure of the elongation factor G. We present data that extend the mimicry hypothesis to initiation factors 1 and 2, originally postulated for the aminoacyl-tRNA x elongation factor Tu x GTP ternary complex, elongation factor G and release factors.

Amino Acid Sequence↗

PubMed1998

Aminoacyl-tRNA analogues; synthesis, purification and properties of 3'-anthraniloyl oligoribonucleotides.

Reaction of isatoic anhydride with adenosine, adenosine 5'-phosphate, oligoribonucleotides or with the E. coli tRNAVal led to attachment of an anthraniloyl residue at 2'- or 3'-OH groups of 3'-terminal ribose residue. No protection of the 5'-hydroxyl group or internal 2'-hydroxyl groups is required for this specific reaction. Anthraniloyl-tRNA which is an analogue of aminoacyl-tRNA forms a ternary complex with EF-Tu*GTP. The anthraniloyl-residue is used as a fluorescent reporter group to monitor interactions with proteins.

Base Sequence↗

PubMed1998

Secondary structure dimorphism and interconversion between hairpin and duplex form of oligoribonucleotides.

RNA hairpins can alternatively form a dimer with a bulged loop flanked by regularly base paired regions. [1H]NMR spectroscopy and native gel electrophoresis were used to study how the sequence of nucleotides in the loop of the hairpin affect the hairpin-duplex interconversion. As a model system, a hairpin containing 7 nucleotides in the loop and 5 base pairs in the stem was used. The loop size was gradually reduced from 7 to 4 nucleotides, yielding finally the stable UNCG tetraloop. Single nucleotide mutations were performed to investigate the influence of the self-complementarity of the loop sequence on the dimerization. The results demonstrate that (1) the initial fraction of hairpin is determined by concentration of the oligonucleotide, the annealing procedure, and the relative stability of the loop, (2) the degree of self-complementarity of the loop sequence of the hairpin governs the dimerization kinetics, and (3) oligonucleotides complementary to the loop sequence decrease the dimerization rate. We propose a secondary structure-based model for the dimerization reaction of RNA hairpins in which the formation of intermolecular base pairs between self-complementary nucleotides of the loops represents the nucleation step.

Base Sequence↗

PubMed1998

The 10Sa RNA gene of Thermus thermophilus.

The 10Sa RNA gene of Thermus thermophilus was isolated and sequenced. The tRNA-like structure at the 5' and 3' ends and other secondary structure features of the T. thermophilus 10Sa RNA are similar to E. coli 10Sa RNA. A variant of the sequence motif coding for the tag peptide is located in the centre of T. thermophilus 10Sa RNA.

Amino Acid Sequence↗

PubMed1997

Interaction of N-tosyl-L-phenylalanylchloromethane with Thermus thermophilus elongation factor Tu.

The interaction of N-tosyl-L-phenylalanylchloromethane (TosPheCH2Cl) with Thermus thermophilus elongation factor Tu (EF-Tu) was studied by affinity labelling and NMR spectroscopy. TosPheCH2Cl binds to GDP and GTP conformers of EF-Tu. The interaction of TosPheCH2Cl with EF-Tu x GDP leads to alkylation of Cys82, while interaction of TosPheCH2Cl with EF-Tu x GTP does not lead to covalent labelling. [A82]EF-Tu, in which the Cys82 is replaced by Ala, has similar properties to wild-type EF-Tu with respect to GTPase activity, binding of guanine nucleotides, interaction with elongation factor Ts (EF-Ts) and interaction with ribosomes. This structural change did not lead to changes, compared with wild-type EF-Tu in the functionality of [A82]EF-Tu, either in the GTP or in the GDP conformation. TosPheCH2Cl binds to EF-Tu x GTP with a dissociation constant of 10 microM. The interaction of TosPheCH2Cl with EF-Tu promotes the hydration of the carbonyl group of TosPheCH2Cl. TosPheCH2Cl competes with aminoacyl-tRNA for its binding site on EF-Tu x GTP. Covalent modification of Cys82 by TosPheCH2Cl does not prevent nucleotide binding and GTPase activity, but interferes with the interaction with aminoacyl-tRNA. TosPheCH2Cl probably mimics the aminoacyl residue of the aminoacyl-tRNA and binds to its binding site on EF-Tu x GTP. This rather specific interaction with EF-Tu x GTP does not allow the modification of Cys82, whereas the loose interaction of TosPheCH2Cl with EF-Tu x GDP leads to alkylation of this residue.

Affinity Labels↗