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B Wittmann-Liebold

Publications and source records attributed to B Wittmann-Liebold.

At least 37 records · Page 2Linked to original sources
PubMed1997

Identification and sequence analysis of contact sites between ribosomal proteins and rRNA in Escherichia coli 30 S subunits by a new approach using matrix-assisted laser desorption/ionization-mass spectrometry combined with N-terminal microsequencing.

Cross-linked peptide-oligoribonucleotide complexes derived from distinct regions of the rRNA and individual ribosomal proteins of the 30 S ribosomal subunits from Escherichia coli were isolated and purified. Cross-linking sites at the amino acid and nucleotide level were determined by N-terminal amino acid sequence analysis in combination with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). MALDI-MS analysis performed subsequent to a partial alkaline hydrolysis of cross-linked peptide-oligoribonucleotide complexes allowed for the first time the cross-linked rRNA moiety to be sequenced by this technique. In this manner Lys-44 in S4 was determined to be cross-linked to the oligoribonucleotide at positions 1531-1542 on the 16 S RNA (whereby either U-1541 or A-1542 is the actual cross-link site), Lys-75 in S7 to positions 1374-1379 (C-1378 cross-linked), Met-114 in S7 to 1234-1241 (U-1240 cross-linked), Lys-55 in S8 to 651-654 (U-653 cross-linked), and Lys-29 in S17 to 629-633 (U-632 cross-linked). The novel approach applied here promises to be useful for similar studies on other known protein.RNA complexes.

Amino Acid Sequence↗
PubMed1997

Identification and characterization of the genes for mitochondrial ribosomal proteins of Saccharomyces cerevisiae.

We have purified 13 large subunit proteins of the mitochondrial ribosome of the yeast Saccharomyces cerevisiae and determined their partial amino acid sequences. To elucidate the structure and function of these proteins, we searched for their genes by comparing our sequence data with those deduced from the genomic nucleotide sequence data of S. cerevisiae and analyzed them. In addition, we searched for the genes encoding proteins whose N-terminal amino acid sequences we have reported previously [Grohmann, L., Graack, H.-R., Kruft, V., Choli, T., Goldschmidt-Reisin, S. & Kitakawa, M. (1991) FEBS Lett. 284, 51-56]. Thus, we were able to identify and characterize 12 new genes for large subunit proteins of the yeast mitochondrial ribosome. Furthermore, we determined the N-terminal amino acid sequences of seven small subunit proteins and subsequently identified the genes for five of them, three of which were found to be new.

Amino Acid Sequence↗
PubMed1997

C-terminal ladder sequencing by an approach combining chemical degradation with analysis by matrix-assisted-laser-desorption ionization mass spectrometry.

A chemical procedure for the degradation of peptides and analysis by matrix-assisted-laser-desorption ionization mass spectrometry (MALDI-MS) was used for C-terminal sequence determination. The method allowed us to determine up to eight amino acid residues in the lower picomole range by mass analysis without any repetitions of the degradation nor any extraction or purification of the truncated peptide chains. The C-terminal degradation of all 20 common amino acid residues was proved by applying this method. Extended C-terminal sequence information from enzymatic digests using carboxypeptidase P was obtained by combining the enzymatic with the chemical mass spectrometric approach. Furthermore the amino acids lysine and glutamine, with the same masses, were distinguishable due to the formation of acetyl-lysine in the chemical process.

Amino Acid Sequence↗
PubMed1997

Phylogenetic relationship of organisms obtained by ribosomal protein comparison.

The evolutionary relationships of ribosomal proteins from eubacteria, archaea, eukaryotes, chloroplasts and mitochondria were examined by their degree of conservation, their structural and functional properties and by the occurrence of conserved structural elements. The structural domains formed by the different protein families were studied. The occurrence of monophyletic groups was investigated for each protein family within the archaea. Phylogenetic trees were constructed between these organisms and together with the homologous sequences of the other phylogenetic domains. All organisms belonging to the archaea clearly formed a monophyletic group. The conserved sequence motifs were checked for the potential to form similar secondary structural elements.

Amino Acid Sequence↗
PubMed1997

Contact sites of peptide-oligoribonucleotide cross-links identified by a combination of peptide and nucleotide sequencing with MALDI MS.

We have investigated peptide-oligoribonucleotide complexes isolated from cross-linked Escherichia coli 30S ribosomal subunits in order to identify the contact sites of these complexes at the molecular level. For this purpose, reversed-phase (RP) HPLC-purified peptide-oligoribonucleotide complexes were submitted to N-terminal amino acid sequencing in order to determine the cross-linked peptide moiety and were analyzed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for calculation of the nucleotide composition of the cross-linked complex. Subsequently, for nucleotide sequence information the complexes were partially hydrolyzed or treated with exonucleases and analyzed again by MALDI-MS. Applying this technique, we were able to identify the cross-linked oligoribonucleotide parts in contact with distinct peptide regions derived from ribosomal proteins S4, S7, S8, and S17 from E. coli.

Amino Acid Sequence↗
PubMed1997

The modular organization of multifunctional peptide synthetases.

Gramicidin S synthetase 2 from B. brevis was affinity labeled at its valine thiolation center with the thiol reagent N-[3H]ethylmaleimide. From a tryptic digest of the enzyme-inhibitor complex a radioactive fragment was isolated in pure form by two reversed-phase HPLC steps. It was identified by liquid-phase N-terminal sequencing in combination with electrospray mass spectrometry (ESI-MS) as a hexadecapeptide containing the thiolation motif LGG(H/D)S(L/I). By ESI-MS it was demonstrated that a 4'-phosphopantetheine cofactor was attached to this fragment at its reactive serine. These results are consistent with the "Multiple Carrier Model" of nonribosomal peptide biosynthesis. Site-specific mutagenesis has been performed in thiolation, elongation, and epimerization motifs of some of the modules of surfactin synthetase from B. subtilis to clarify the function of prominent conserved amino acid residues in the intermediate steps of peptide biosynthesis. The modular structure of multifunctional peptide synthetases is discussed.

Amino Acid Isomerases↗
PubMed1996

Characterization of the human small-ribosomal-subunit proteins by N-terminal and internal sequencing, and mass spectrometry.

Reverse-phase HPLC was used to fractionate 40S ribosomal proteins from human placenta. Application of a C4 reverse-phase column allowed us to obtain 27 well-resolved peaks. The protein composition of each chromatographic fraction was established by two-dimensional polyacrylamide gel electrophoresis and N-terminal sequencing. N-terminally blocked proteins were cleaved with endoproteinase Lys-C, and suitable peptides were sequenced. All sequences were compared with those of ribosomal proteins available from data bases. This allowed us to identify all proteins from the 40S human ribosomal subunit in the HPLC elution profile. By matrix-assisted laser-desorption ionization mass spectrometry the masses of the 40S proteins were determined and checked for the presence of post-translational modifications. For several proteins differences to the deduced sequences and the calculated masses were found to be due to post-translational modifications.

Amino Acid Sequence↗
PubMed1996

The multiple carrier model of nonribosomal peptide biosynthesis at modular multienzymatic templates.

Gramicidin S synthetase 1 and 2 were affinity-labeled at their thiolation centers either by thioesterification with the amino acid substrate or by specific alkylation with the thiol reagent N-ethylmaleimide in combination with a substrate protection technique. The labeled proteins were digested either chemically by cyanogen bromide or by proteases. An efficient multistep high pressure liquid chromatography methodology was developed and used to isolate the active site peptide fragments of all five thiolation centers of gramicidin S synthetase in pure form. The structures of these fragments are investigated by N-terminal sequencing, mass spectrometry, and amino acid analysis. Each of the active site peptide fragments contains the consensus motif LGG(H/D)S(L/I), which is specific for thioester formation in nonribosomal peptide biosynthesis. It was demonstrated that a 4'-phosphopantetheine cofactor is attached to the central serine of the thiolation motif in each amino acid-activating module of the gramicidin S synthetase multienzyme system forming the thioester binding sites for the amino acid substrates and catalyzing the elongation process. Our data are strong support for a "multiple carrier model" of nonribosomal peptide biosynthesis at multifunctional templates, which is discussed in detail.

Amino Acid Isomerases↗
PubMed1996

Identification of human myocardial proteins separated by two-dimensional electrophoresis using an effective sample preparation for mass spectrometry.

Peptide mass fingerprinting is a powerful tool for the identification of proteins separated by two-dimensional gel electrophoresis (2-DE). The identification of in-gel digested proteins by peptide mass fingerprinting was significantly improve in comparison to blot-digests by using a peptide-collecting device. This device allows the effective purification and concentration of enzymatic digests of low-intensity spots without expensive equipment and is described in detail. Sensitivity in the fmol range was demonstrated by unequivocal identification of bovine serum albumin after sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Furthermore the high performance liquid chromatography pattern of in-gel digests indicated a 2- to 3-fold higher yield of the separated peptides. Therefore, a higher amount of the peptides was available to perform N-terminal sequencing. The identification of 16 proteins from a high-resolution 2-DE gel map of human myocardium tissue has been achieved by means of this technique. Three of these proteins were associated with changes in spot intensity with dilated cardiomyopathy.

Acetyl-CoA C-Acyltransferase↗
PubMed1996

High-performance human myocardial two-dimensional electrophoresis database: edition 1996.

The master gel of the human myocardial two-dimensional electrophoresis (2-DE) gel database contains about 3300 protein spots characterized in terms of isoelectric point (pI) and molecular mass. A high-performance technique was applied, using large gels (23 x 30 cm). Isoelectric focusing with anodic sample preparation and nonequilibrium running conditions (NEPHGE) was combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 15% acrylamide gels in the second dimension. The range of pI extends from pH 4.5 to 9.6. Seventy proteins were identified by combinations of amino acid analysis, N-terminal and internal sequencing, immunostaining, matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) peptide mass fingerprinting, post-source decay MALDI-MS and ladder sequencing by carboxypeptidase P. The identification of additional proteins, not found in the master gel, was achieved by immunoblotting. Unequivocal identification with high sensitivity and good yield was obtained by combining internal sequencing and MALDI-MS. In-gel digestion, the concentration and purification of peptides in a peptide collecting device, and the improved FRAGMOD program for peptide mass fingerprinting have added to the security and sensitivity of identification. The high-performance human myocardial 2-DE database was built up with proteins detected by the TOPSPOT program. Spots within six sections of the whole pattern are clickable. Protein description includes detailed information about identification, characterization, and links to the related SWISS-PROT, other 2-DE databases and Medline entries. The database is constructed in accordance with four of the rules for a federated database.

Computer Communication Networks↗
PubMed1995

Partial amino acid sequence of an L-amino acid oxidase from the cyanobacterium Synechococcus PCC6301, cloning and DNA sequence analysis of the aoxA gene.

A novel type of L-amino acid oxidase from Synechococcus PCC6301 was purified and subjected to amino acid sequence analysis. Since the N-terminus of the L-amino acid oxidase protein was not accessible for Edman degradation, the protein was partially hydrolysed and a contiguous sequence of 17 amino acid residues was obtained from an endogenous peptide fragment. Based on the partial peptide sequence two oligonucleotides were designed, which were used as probes in Southern hybridization experiments in order to identify the corresponding aoxA gene. The aoxA gene was isolated from a size-fractionated genomic library of Synechococcus PCC6301 and subsequently sequenced. From the nucleotide sequence (data base accession number Z48565) it can be deduced that the L-amino acid protein consists of 355 amino acid residues resulting in a molar mass of 39.2 kDa. The calculated isoelectric point of the protein is 9.81. The L-amino acid oxidase from Synechococcus PCC6301 shows low homologies to other flavin oxidases/dehydrogenases, especially amine oxidases, but no homologies to other so far sequenced L- or D-amino acid oxidases.

Amino Acid Oxidoreductases↗
PubMed1995

Cartography of ribosomal proteins of the 30S subunit from the halophilic Haloarcula marismortui and complete sequence analysis of protein HS26.

By two-dimensional polyacrylamide gel electrophoresis of 30S ribosomal subunit proteins (S proteins) from Haloarcula marismortui we identified 27 distinct spots and analyzed all of them by protein sequence analysis. We demonstrated that protein HmaS2 (HS2) is encoded by the open reading frame orfMSG and has sequence similarities to the S2 ribosomal protein family. The proteins HmaS5 and HmaS14 were identified as spots HS7 and HS21/HS22, respectively. Protein HS4 was characterized by amino-terminal sequence analysis. The spot HS25 was recognized as an individual protein and also characterized by sequence analysis. Furthermore, the complete primary sequence of HS26 is reported, showing similarity only to eukaryotic ribosomal proteins. The sequence data of a further basic protein shows a high degree of similarity to ribosomal protein S12, therefore, it was designated HmaS12. Slightly different results compared to published sequence data were obtained for the protein HS12 and HmaS19. The putative 'ribosomal' protein HSH could not be localized in the two-dimensional pattern of the total 30S ribosomal subunit proteins of H. marismortui. Therefore, it seems to be unlikely that this protein is a real constituent of the H. marismortui ribosome.

Amino Acid Sequence↗
PubMed1995

Peptide environment of the peptidyl transferase center from Escherichia coli 70 S ribosomes as determined by thermoaffinity labeling with dihydrospiramycin.

In an attempt to gain information about the peptidyl transferase center at the peptide level we cross-linked the spiramycin derivative dihydrospiramycin to its functional binding site in the 70 S ribosome of Escherichia coli. In this manner ribosomal proteins S12, S14, L17, L18, L27 and L35 were found specifically affinity-labeled. Proteolytic fragmentation of these proteins, separation by C18 reversed-phase high performance liquid chromatography of the peptide mixtures, and subsequent sequence analysis of labeled peptides revealed peptide regions at positions Ala1-Lys9 and Tyr116-Lys119 of S12, Leu47-Asp53 of protein S14, Ser6-Lys35 of protein L17, Ala57-Lys63 of protein L18, Ala5-Lys18 and Val66-Lys71 of protein L27, and Thr5-Lys11 of protein L35. This approach is a valuable tool to characterize the binding site of spiramycin as well as the peptidyl transferase center at the molecular level.

Affinity Labels↗
PubMed1995

Protein-rRNA binding features and their structural and functional implications in ribosomes as determined by cross-linking studies.

We have investigated protein-rRNA cross-links formed in 30S and 50S ribosomal subunits of Escherichia coli and Bacillus stearothermophilus at the molecular level using UV and 2-iminothiolane as cross-linking agents. We identified amino acids cross-linked to rRNA for 13 ribosomal proteins from these organisms, namely derived from S3, S4, S7, S14, S17, L2, L4, L6, L14, L27, L28, L29 and L36. Several other peptide stretches cross-linked to rRNA have been sequenced in which no direct cross-linked amino acid could be detected. The cross-linked amino acids are positioned within loop domains carrying RNA binding features such as conserved basic and aromatic residues. One of the cross-linked peptides in ribosomal protein S3 shows a common primary sequence motif--the KH motif--directly involved in interaction with rRNA, and the cross-linked amino acid in ribosomal protein L36 lies within the zinc finger-like motif of this protein. The cross-linked amino acids in ribosomal proteins S17 and L6 prove the proposed RNA interacting site derived from three-dimensional models. A comparison of our structural data with mutations in ribosomal proteins that lead to antibiotic resistance, and with those from protein-antibiotic cross-linking experiments, reveals functional implications for ribosomal proteins that interact with rRNA.

Amino Acid Sequence↗
PubMed1995

Protein analysis on a genomic scale.

Methods for protein analysis, such as chromatography, electrophoresis, enzyme tests, receptor assays and immunological tests, have always been aimed in a classical reductionistic manner at investigating single proteins isolated from the complex protein composition of biological compartments. The complexity of the protein composition in biological systems was first visualized by two-dimensional electrophoresis (2-DE). Using 2-DE like a molecular microscope, protein variations between different biological situations may be detected by subtractive 2-DE analyses. Combining 2-DE with microsequencing, amino acid analysis and mass spectrometry protein spots on 2-DE gels may be identified. The sequence information can be used to find the gene. However, by 2-DE not only single protein changes can be detected and investigated on the gene level, but also complex changes of many proteins on a genomic scale.

Amino Acids↗
PubMed1995

Gramicidin S synthetase 1 (phenylalanine racemase), a prototype of amino acid racemases containing the cofactor 4'-phosphopantetheine.

The biosynthesis of the decapeptide antibiotic gramicidin S in Bacillus brevis ATCC 9999 is catalyzed by a multienzyme system consisting of two multifunctional proteins, gramicidin S synthetase 1 and 2, encoded by the grsA and grsB genes, respectively. Gramicidin S synthetase 1 (phenylalanine racemase, EC 5.1.1.11, GS1) racemizes phenylalanine in the thioester-bound stage. The amount of 4'-phosphopantetheine liberated from highly purified GS1 was determined microbiologically using Lacto-bacillus plantarum as the test organism. It matches exactly with the amount of L-[14C]phenylalanine covalently incorporated by GS1 as thioester. The reaction center of GS1 for L-phenylalanine thiolation and racemization was labeled with [3H]iodoacetic acid. After tryptic fragmentation of the 3H-carboxymethylated enzyme, the active site peptide for thioester binding and racemization of phenylalanine was isolated in pure form by multistep methodology and investigated by sequence, amino acid, and mass spectrometric analysis. A 4'-phosphopantetheine carrier was found to be attached to the active site serine of the consensus motif LGGDSI forming the thiolation site of phenylalanine. These specific properties establish GS1 as a prototype of amino acid racemases using 4'-phosphopantetheine as a cofactor and yield further evidence that multiple Pan carriers are involved in gramicidin S formation. Our results are strong evidence for the "multiple carrier model" as a new concept of nonribosomal peptide biosynthesis at protein templates as recently proposed [Stein, T., et al. (1994) FEBS Lett. 340, 39-44].

Amino Acid Isomerases↗
PubMed1995

MALDI-MS for C-terminal sequence determination of peptides and proteins degraded by carboxypeptidase Y and P.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used for C-terminal amino acid sequence determination of peptides and proteins. The usefulness of MALDI-MS was demonstrated by analyzing peptide mixtures (C-terminal peptide ladder) which were generated by enzymatic digestion of substance P, glucagon, angiotensinogen, insulin B chain and myoglobin with the exopeptidases carboxypeptidase Y and P. The results clearly show that up to 11 amino acid residues can be determined in the pmol range by analyzing the molecular masses of the truncated peptides. For proteins it is possible to investigate enzymatic or chemical digests in the same manner.

Amino Acid Sequence↗