Biomedical subjects

B Wittmann-Liebold

Publications and source records attributed to B Wittmann-Liebold.

At least 55 records · Page 3Linked to original sources

PubMed1995

Amino acid sequence of the ribosomal protein HS23 from the halophilic Haloarcula marismortui and homology studies to other ribosomal proteins.

The ribosomal protein HS23 from the 30S subunit of the extreme halophilic Haloarcula marismortui, belonging to the group of archaea, was isolated either by RP-HLPLC or two-dimensional polyacrylamide gel electrophoresis. The complete amino acid sequence was determined by automated N-terminal microsequencing. The protein consists of 123 residues with a corresponding molecular mass of 12,552 Da as determined by electrospray mass spectroscopy; the pI is 11.04. Homology studies reveal similarities to the eukaryotic ribosomal protein S8 from Homo sapiens, Rattus norvegicus, Leishmania major, and Saccharomyces cerevisiae.

Amino Acid Sequence↗

PubMed1995

Determination of peptide regions exposed at the surface of the bacterial ribosome with antibodies against synthetic peptides.

We synthesized six peptides corresponding to regions that are predicted to be surface-exposed of the following ribosomal proteins: protein L2, positions (D263-K272); protein L5, positions (I136-G150); protein L25, positions (Q75-D90); protein S3, positions (Q222-K232) derived from Escherichia coli; and protein L2, positions (K257-K275), and protein S3, positions (R130-T150) from Bacillus stearothermophilus. These peptides were employed to raise ribosomal protein-cross-reactive antibodies. The anti-peptide antisera reacted specifically with their parent proteins, as demonstrated by immunoblotting experiments. In a competition assay proteins L2 from E. coli and B. stearothermophilus as well as proteins L5 and L25 from E. coli were found to be accessible to the respective anti-peptide antibodies in the 50S subunits, but not in 70S ribosomes, proving their location at the 50S interface which is covered by the 30S subunit in the 70S complex. Two of the anti-peptide antisera directed against sequences deduced from protein S3 of E. coli and B. stearothermophilus reacted with 30S subunits as well as with 70S ribosomes, demonstrating their location at the backside, which is exposed to solvent. Thus, by the strategy applied specific short peptide stretches were located at the surface of the ribosome.

Amino Acid Sequence↗

PubMed1994

Complete amino acid sequence of ribosomal protein S14 from Bacillus stearothermophilus and homology studies to other ribosomal proteins.

The complete amino acid sequence of protein S14 from the small subunit of Bacillus stearothermophilus was determined by N-terminal sequence analysis and by sequencing of overlapping peptides obtained from enzymatic digestions. Protein S14 consists of 60 amino acid residues with a molecular mass of 7148 Da. It has a high content of basic amino acids and a predicted isoelectric point of 11.46. Protein S14 contains two pairs of cysteines in the carboxyl-terminal region, presumably linked by two sulphur bridges. A comparison between protein S14 of B. stearothermophilus and homologous proteins from other organisms revealed highly conserved carboxyl-termini for this protein in eubacteria, archaebacteria and eukaryotes.

Amino Acid Sequence↗

PubMed1994

Analysis of the puromycin binding site in the 70 S ribosome of Escherichia coli at the peptide level.

Photoinduced cross-linking of Escherichia coli 70 S ribosomes with [3H]puromycin has led to the labeling of ribosomal proteins S7, S14, S18, L18, and L29. Proteolytic fragmentation of these proteins and separation of the peptide mixtures by C18 reversed-phase high performance liquid chromatography resulted in six puromycin-labeled peptides which were applied to sequence analysis. The following peptides were found labeled: Pro1-Lys10 of S7, Ala28-Lys46 and Ala7-Lys11 of S14, Asp24-Lys29 of S18, Tyr64-Lys68 of L18, and Thr55-Lys60 of L29. For the first time the molecular environment of an antibiotic in the procaryotic ribosome is presented at the peptide level.

Amino Acid Sequence↗

PubMed1994

Comparative analysis of the protein components from 5S rRNA.protein complexes of halophilic archaebacteria.

The 5S RNA.protein complexes have been isolated from the 50S subunit of the halophilic archaebacteria Halobacterium cutirubrum, Halobacterium halobium, Halobacterium salinarium, Haloferax mediterranei, Haloferax volcanii and Haloarcula marismortui. The 50S subunits from most of the halophiles released a multiprotein ribonucleoprotein particle similar to that previously observed with the H. cutirubrum 5S RNA.protein complex, which contained proteins from the L5 and L18 ribosomal protein families. Ribosomes from H. marismortui, however, released an RNA.protein complex containing a single protein (L18) that is homologous to the single protein found in the eukaryotic 5S ribonucleoprotein complexes. N-terminal sequence analyses of the halophilic 5S RNA-binding proteins suggest that the L18 protein primary structure is highly conserved, with only the H. marismortui protein having a sequence difference in at least the first twenty amino acids. Although the L5 group of ribosomal proteins also shows a high conservation, it appears that the proteins may have had more freedom to diverge throughout evolution.

Amino Acid Sequence↗

PubMed1994

Detection of 4'-phosphopantetheine at the thioester binding site for L-valine of gramicidinS synthetase 2.

Biosynthesis of gramicidinS in Bacillus brevis is catalysed by a multienzyme system consisting of two multifunctional proteins, gramicidinS synthetase 1 and 2 codified by the grsA and grsB genes, respectively. GramicidinS synthetase 2 shows a modular architecture of four amino acid-activating domains each containing a thioester binding motif LGG H/D S L/I highly conserved in its C-terminal region, as demonstrated by sequence analysis of the grsB gene [W. Schlumbohm et al. (1991) J. Biol. Chem. 266, 23135-23141]. This multienzyme was specifically labeled at the thioester binding site of L-valine with [3H]N-ethylmaleimide using a substrate protection technique. After enzymatic digestion a labeled active site peptide was isolated in pure form by multistep methodology. This fragment was identified by gas-phase sequencing as the active site peptide of the thiotemplate site for L-Val by comparison with the grsB gene sequence. By mass spectrometry in combination with amino acid analysis it was demonstrated that a 4'-phosphopantetheine carrier was attached to the active serine in this motif. Our results give evidence that multiple peripheral 4'-phosphopantetheine carriers are involved in the formation of gramicidinS in contrast to a central carrier arm as assumed in the original version of the thiotemplate mechanism. A 'Multiple Carrier Model' of nonribosomal peptide biosynthesis is proposed.

Amino Acid Isomerases↗

PubMed1994

Protein composition of the human heart: the construction of a myocardial two-dimensional electrophoresis database.

Molecular changes occurring in myocardial diseases are not well understood. Proteins, as regulatory molecules, should play an important role in the etiology of these diseases. The method of two-dimensional electrophoresis (2-DE) allows the analysis of some thousand proteins with one experiment. An important prerequisite for this kind of investigation is the possibility of identifying the proteins separated by 2-DE. We resolve 3239 proteins of the human myocardium and tried to identify 33 proteins by amino acid analysis and microsequencing. Twenty proteins were identified by search for the protein-chemical data obtained in the Martinsried Institute Protein Sequence Database. Comparisons of 2-DE patterns of different size, which were obtained in different laboratories, were performed with the result that proteins identified on a 2-DE map of one laboratory can be assigned to spots of 2-DE maps produced by another laboratory. Our results show the usefulness of a myocardial 2-DE database; they can be used in different laboratories and make it possible to generate a collection of important human myocardial proteins in a 2-DE database for comparative studies worldwide.

Amino Acid Sequence↗

PubMed1993

Localization of proteins HL29 and HL31 from Haloarcula marismortui within the 50 S ribosomal subunit by chemical crosslinking.

Isolated 50 S ribosomal subunits from the halophilic archaebacterium Haloarcula marismortui were treated in situ with the homobifunctional and cleavable crosslinking reagent dithiobis(succinimidyl propionate) (12 A). Several crosslinked complexes were obtained. Among these were the protein pairs HmaL4-HL29 and HmaL18-HL31; HL29 and HL31 are ribosomal proteins without any equivalent in eubacterial ribosomes. The crosslinked protein pairs were isolated on a preparative scale by combining conventional ion-exchange chromatography and reverse phase high-pressure liquid chromatography. The monomeric proteins involved in crosslink formation were unambiguously identified by two-dimensional gel electrophoresis and N-terminal or internal protein sequencing. Due to the homology between HmaL4 and HmaL18 and their Escherichia coli counterparts, and the roughly known location of these proteins within the 50 S subunit, our results demonstrate that HL29 is probably located in the centre of the large subunit in the vicinity of the peptidyltransferase domain, whereas HL31 must be situated within the central protuberance close to the region of the 5 S RNA.

Amino Acid Sequence↗

PubMed1993

HL35e and HLA: primary structure of two very basic and cysteine-rich ribosomal proteins from Haloarcula marismortui.

Two small and very basic ribosomal proteins have been purified from the 50S ribosomal subunit of the archaebacterium Haloarcula marismortui by RP-HPLC. The complete primary structures of these two proteins, which we refer to as HL35e and HLA, have been determined by protein chemical methods. Both proteins are characterized by a high content of basic amino acids and the presence of two pairs of cysteines in each polypeptide chain, one of which resembles the C4-zinc-finger motif. Comparison of the protein sequences with those of other ribosomal proteins revealed that HL35e shows significant sequence homology exclusively to eukaryotic ribosomal proteins, namely to yeast L35 and to L37 from rat. For HLA no homologous ribosomal protein so far known could be found. Obviously, HL35e and HLA have no counterparts in eubacterial ribosomes.

Amino Acid Sequence↗

PubMed1993

Identification of cross-linked amino acids in the protein pair HmaL23-HmaL29 from the 50S ribosomal subunit of the archaebacterium Haloarcula marismortui.

50S ribosomal subunits from the extreme halophilic archaebacterium Haloarcula marismortui were treated with the homobifunctional protein-protein cross-linking reagents diepoxybutane (4 A) and dithiobis(succinimidyl propionate) (12 A). The dominant product with both cross-linking reagents was identified on the protein level as HmaL23-HmaL29, which is homologous to the protein pair L23-L29 from Escherichia coli [Walleczek, J., Martin, T., Redl, B., Stöffler-Meilicke, M., & Stöffler, G. (1989) Biochemistry 28, 4099-4105] and from Bacillus stearothermophilus [Brockmöller, J., & Kamp, R. M. (1986) Biol. Chem. Hoppe-Seyler 367, 925-935]. To reveal the exact cross-linking site in HmaL23-HmaL29, the cross-linked complex was purified on a preparative scale by conventional and high-performance liquid chromatography. After endoproteolytic fragmentation of the protein pair, the amino acids engaged in cross-link formation were unambiguously identified by N-terminal sequence analysis and mass spectrometry of the cross-linked peptides. The cross-link is formed between lysine-57 in the C-terminal region of HmaL29 and the alpha-amino group of the N-terminal serine in protein HmaL23, irrespective of the cross-linking reagent. This result demonstrates that the N-terminal region of protein HmaL23 and the C-terminal domain of HmaL29 are highly flexible so that the distance between the two polypeptide chains can vary by at least 8 A. Comparison of our cross-linking results with those obtained with B. stearothermophilus revealed that the fine structure within this ribosomal domain is at least partially conserved.

Archaea↗

PubMed1993

Cross-linked amino acids in the protein pairs L3-L19 and L23-L29 of Bacillus stearothermophilus ribosomes after treatment with diepoxybutane.

Treatment of native 50 S ribosomal subunits of Bacillus stearothermophilus with the homobifunctional cross-linking reagent diepoxybutane generated two cross-linked protein pairs, L3-L19 and L23-L29, which were isolated and identified. The analysis of the cross-linking sites at the amino acid level in both protein pairs is presented. Using a combination of sequence analysis and mass spectrometry it could be demonstrated that His-28 in protein L3 and the N-terminal amino acids Met-1, His-2, and His-3 in protein L19 are involved in forming the cross-link L3-L19. Within the pair L23-L29 Met-1 in protein L23 and Lys-4 in protein L29 were identified as cross-linking sites employing a similar approach. Comparison of our data with results derived from other cross-linking experiments showed that in general the structural organization of the ribosomes in eubacteria (the Gram-positive B. stearothermophilus and the Gram-negative Escherichia coli) has been conserved to quite an extent during evolution but that the fine structures differ slightly. By mass spectrometry the specificity of diepoxybutane and its cleaving mechanism using sodium periodate could be examined. In addition the complete amino acid sequence of protein L19 of B. stearothermophilus has been determined and revealed 58% identical amino acid residues to the homologous E. coli protein L19.

Amino Acid Sequence↗

PubMed1993

N-terminal modification and amino-acid sequence of the ribosomal protein HmaS7 from Haloarcula marismortui and homology studies to other ribosomal proteins.

The ribosomal protein HmaS7 from the 30S subunit of the extreme halophilic archaeum Haloarcula marismortui was isolated by semi-preparative RP-HPLC. The complete amino-acid sequence of this protein was determined by automated microsequence analysis of appropriate peptide fragments from several proteinase digests. The entire protein consists of 205 amino acids with a corresponding molecular mass of 22580 Da. The modification at the amino-terminal amino acid was deblocked so that the N-terminal amino acids could be sequenced and the type of the modification was identified as an acetyl group by electrospray mass spectrometry of suitable peptides. Homology studies of HmaS7 showed similarities to ribosomal proteins derived from organisms of all three urkingdoms, such as to EcoS7, HmoS7, MvaS7, SacS7 and RatS7; due to the strong sequence homologies found within the archaebacterial ribosomal proteins we conclude that the protein sequence which was determined for S7 from Methanococcus vannielii by nucleotide sequencing of the gene should be about 20 or 30 amino acids longer than previously published (Lechner, K., Heller, G. & Böck, A. (1989) J. Mol. Evol. 29, 20-27).

Amino Acid Sequence↗

PubMed1993

Isolation and characterization of a new ribosomal protein from the thermophilic eubacteria, Thermus thermophilus, T. aquaticus and T. flavus.

A ribosomal protein, showing no homology with other known prokaryotic ribosomal proteins, was isolated and characterized from the thermophilic eubacteria, Thermus thermophilus, T. aquaticus and T. flavus. This small (26 amino acids) and strongly basic (1 acidic and 13 basic residues) protein displayed the same primary structure from all three sources. Interestingly, it shows about 65% homology with a ribosomal protein from spinach chloroplasts (J. Schmidt, personal communication).

Amino Acid Sequence↗

PubMed1992

Primary structures of ribosomal proteins L3 and L4 from Bacillus stearothermophilus.

Ribosomal proteins L3 and L4 were purified to homogeneity from total protein isolated from the 50S subunit of Bacillus stearothermophilus by reversed-phase high-performance liquid chromatography (RP-HPLC). Amino acid sequences of both proteins were determined by automated N-terminal sequence analysis and sequencing of internal peptides. Using oligonucleotides deduced from the N-terminal region of protein L3 as hybridization probes, a DNA fragment coding for proteins L3, L4 and the N-terminal part of protein L23 has been identified, cloned and sequenced. The organization of the genes is identical to that found in the S10 operon of Escherichia coli. Comparison of the sequences of proteins L3 and L4 with those of other organisms revealed that all proteins of the L3 family are highly conserved. On the other hand, the archaebacterial L4 proteins show no significant sequence similarity to the E. coli L4 protein whereas the L4 protein of B. stearothermophilus is significantly similar to all of the L4 proteins and thus justifies the membership of all the L4 proteins in one protein family. The results are discussed with respect to the phylogenetic relationship between eubacteria, archaebacteria and eukaryotes and possible functional domains of proteins L3 and L4.

Amino Acid Sequence↗

PubMed1991

Determination of peptide regions on the surface of the eubacterial and archaebacterial ribosome by limited proteolytic digestion.

Limited proteolysis was used in combination with two-dimensional gel electrophoresis, blotting, and amino acid sequence analysis to investigate the surface of intact ribosomal subunits at the peptide and amino acid level. Surface sites of 14 ribosomal proteins from Escherichia coli 50S subunits were determined using proteases with different specificities. To assess the evolutionary conservation of ribosomal topography among eubacteria, large subunits from Bacillus stearothermophilus were also subjected to limited proteolysis. The results obtained indicate a conservation of the three-dimensional ribosomal structure at the peptide level. The data for the eubacterial ribosomes are in full agreement with the model of the 50S protein topography derived from immunological data. Furthermore, peptide surface regions of archaebacterial ribosomes have been investigated. The results presented in this work prove that limited proteolysis can successfully be applied to halophilic and thermophilic ribosomes from archaebacteria.

Amino Acid Sequence↗

PubMed1991

An active serine is involved in covalent substrate amino acid binding at each reaction center of gramicidin S synthetase.

The condensing peptide forming multienzyme of gramicidin S synthetase (gramicidin S synthetase 2) was specifically labeled at its putative thiotemplate sites for L-valine and L-leucine by covalent incorporation of the 14C-labeled substrate amino acids. The thioester complexes of the multienzyme were digested with CNBr, Staphylococcus aureus V8 protease, and pepsin. Reaction center peptides containing the [14C]valine and [14C]leucine labels were isolated in pure form. They show a high degree of sequence similarity and contain the same consensus sequence LGGH/DXL. The labels were eliminated in the first Edman degradation step. A dehydroalanine was identified which can originate from either a cysteine or a serine. The comparison of the chemical results with the deduced amino acid sequence of the grsB gene encoding the gramicidin S synthetase 2 revealed that 4 such motifs are located within the gene structure, each of them being localized in the 3'-terminal region of one of 4 gene segments grsB1-B4. They have a size of approximately 2 kilobases and presumably code for the 4 amino acid activating domains of the synthetase. Surprisingly a serine was found at each putative substrate amino acid-binding position instead of a cysteine as postulated by the thiotemplate mechanism. Therefore the data suggest that active serine residues are involved in nonribosomal peptide syntheses of microbial peptides.

Amino Acid Isomerases↗

PubMed1991

On-sequencer pyridylethylation of cysteine residues after protection of amino groups by reaction with phenylisothiocyanate.

Cysteine residues in polypeptides are not easily identified during automated N-terminal sequence analysis. Reaction of cysteine side chains with 4-vinylpyridine and identification as the pyridylethylated phenylthiohydantion derivative (PE-PTH-Cys) were proposed. However, after this reaction a desalting step is necessary. If limited sample amounts do not allow this desalting step, on-sequencer pyridylethylation is an alternative, although preview of the consecutive amino acid is usually observed in this case. We describe an on-sequencer procedure that avoids such preview formation by derivatizing the peptide with phenylisothiocyanate (PITC) prior to reaction with 4-vinylpyridine. The pyridylethylation is performed in the cartridge of the sequencer after immobilization of the protein or peptide on a polybrene-coated glass fiber filter and thiocarbamylation with PITC. Preview caused by N-alkylation is not observed and PE-PTH-Cys is detected in much higher yields than usual. The procedure reported here is significantly shortened, optimized to reduce side products, and avoids losses during sample handling. It can easily be adapted to any automated version of the sequencers.

Amino Acid Sequence↗

PubMed1991

Microsequencing of proteins and peptides in the Knauer sequencer with and without covalent attachment to polyvinylidene difluoride membranes by the wet-phase degradation technique.

Proteins and large peptides were degraded with phenylisothiocyanate (PITC) in the horizontal flow-through-reactor of the Modular Knauer Sequencer (Fischer, S., Reimann, F. & Wittmann-Liebold, B. (1989) in Methods in Protein Sequence Analysis (Wittmann-Liebold, B., ed.) Springer-Verlag, Berlin, pp. 98-107) by the wet-phase filter technique (Wittmann-Liebold, B. (1988) J. Prot. Chem. 7, 224-225) employing polyvinylidene difluoride (PVDF) membranes without polybrene. In order to prevent losses of small peptides during solvent washes at the degradation, 1.4-phenylene diisothiocyanate (DITC) derivatized PVDF support (MilliGen, Burlington, MA) was used to covalently attach the peptide via its lysine groups in situ within the cross-flow reaction chamber onto this membrane (Herfurth, E., Pilling, U. & Wittmann-Liebold, B. (1990) J. Prot. Chem. 9, 267). We found these membranes very suitable for peptide degradations in the Knauer sequencer. In almost all cases we were able to identify the amino-acid residues of the peptide up to its last covalent fixation point to the membrane.

Amino Acid Sequence↗