Biomedical subjects

B Wittmann-Liebold

Publications and source records attributed to B Wittmann-Liebold.

At least 19 recordsLinked to original sources

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↗

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↗

PubMed1991

The amino-acid sequences of the Bacillus stearothermophilus ribosomal proteins S17 and S21 and their comparison to homologous proteins of other ribosomes.

Ribosomal proteins S17 and S21 from the moderate thermophile Bacillus stearothermophilus were purified by one-step high-performance liquid chromatography from the 30S-subunit protein mixture employing a semi-preparative reversed-phase C4 column. The complete amino-acid sequences of these proteins were determined by a combination of N-terminal sequencing in picomole quantities of the protein and of appropriate peptide fragments. Proteins S17 and S21 consist of 86 and 55 amino-acid residues, corresponding to molecular masses of 10074 and 6593 Da, respectively. They are homologous to proteins S17 and S21 from the Escherichia coli ribosome, showing 50 and 55% identities in the corresponding regions, respectively. The C-terminal region of protein S21 from B. stearothermophilus has a deletion of 15 residues as compared to the E. coli S21 protein. The evolutionary relationships of the Bacillus proteins to various other members of the S17 and S21 ribosomal protein families are discussed.

Amino Acid Sequence↗

PubMed1990

Protein blotting followed by microsequencing.

The use of new membranes such as activated or derivatized glass fibers as well as synthetic membranes, which are compatible with the hazardous sequencing reagents, are described. Precautions to be taken in order to prevent N-terminal blockage of the proteins during electrophoresis and blotting are described, as well as the conditions for protein detection after blotting and protein treatment for in situ amino acid analysis, fragmentation and microsequencing. For a number of standard proteins and bacterial ribosomal proteins microsequence analysis is reported for two commercially available sequencers (Applied Biosystems and Knauer).

Amino Acid Sequence↗

PubMed1990

Processing of pseudorabies virus glycoprotein gII.

The glycoprotein complex gII of pseudorabies virus was isolated by immunoprecipitation with the monoclonal antibody M5, which was covalently linked to protein A-Sepharose. After sodium dodecyl sulfate-polyarylamide gel electrophoresis under reducing conditions and blotting onto poly(vinylidene difluoride) membrane, its subunits, gIIa, gIIb, and gIIc, were subjected to N-terminal sequencing. gIIa and gIIb start at position 59 and gIIc starts at position 503 according to the amino acid sequence deduced from the gene, indicating that there is one major protein (gIIa) which is cleaved into the two protein fragments gIIb and gIIc. Protein labeling with 14C-amino acids gave no indication that the three proteins (gIIa, gIIb, and gIIc) of the complex are present in equimolar ratios. It seems that gIIa is only a minor component of the complex, whereas gIIb and gIIc are contained in equimolar amounts.

Amino Acid Sequence↗

PubMed1990

Use of a hapten specific anti-dansyl antibody for the localization of ribosomal proteins by immuno electron microscopy.

The fluorescent reagent dansyl chloride has been used as an immunological marker for the electron microscopic localization of ribosomal proteins on the surface of 50S ribosomal subunits. The proteins BstL1 from Bacillus stearothermophilus and EcoL1 from Escherichia coli were dansylated to various degrees and reconstituted into the L1-deficient E. coli 50S subunits from mutant MV17-10. Using antibodies specific to dansyl chloride, both proteins were mapped at the lateral protuberance near the peptidyl transferase center.

Antibody Specificity↗

PubMed1989

Blotting of proteins onto Immobilon membranes. In situ characterization and comparison with high-performance liquid chromatography.

The electrophoretic transfer from polyacrylamide gels to Immobilon [poly(vinylidene difluoride)] membranes of various proteins differing in molecular masses from 14,000 to 200,000 was performed, using both a semi-dry blotting apparatus and a standard blotting chamber. The blotted proteins were analyzed and sequenced with and without staining, and the initial yields of the degradation were examined. Furthermore, protein purification by blotting after one- and two-dimensional gel electrophoresis was compared with conventional HPLC methods. Optimum blotting conditions for in situ enzymatic or chemical cleavages of the proteins on the blots are described, and for the in situ hydrolysis followed by amino acid analysis and cysteine determination.

Blotting, Western↗

PubMed1989

Sequence similarity between protein B and human apolipoprotein A-IV.

Sequence comparison of protein B (CAMP-factor) with human apolipoprotein A-IV (apo A-IV) revealed 32% similarity between the N-terminal part of protein B and a part of the putative lipid-binding domain of apo A-IV. The significance of this similarity is discussed with respect to the structure/function relationship of protein B.

Amino Acid Sequence↗

PubMed1989

An archaebacterial gene from Methanococcus vannielii encoding a protein homologous to the ribosomal protein L10 family.

An open reading frame upstream of the Methanococcus vannielii L12 gene has been detected. The beginning of this open reading frame agrees with the N-terminal region of a protein (MvaL10) which has been isolated from the 50 S ribosomal subunit of M. vannielii and sequenced. The length of this gene is 1008 nucleotides, coding for 336 amino acids. Excellent sequence similarities were found to the L10-like ribosomal proteins from Halobacterium halobium and man. The N-terminal part of the MvaL10 protein shows significant sequence similarities to the E. coli L10 protein. MvaL10 is more than twice as long as E. coli L10 but is of length similar to those of the homologous halobacterial and human proteins. Interestingly, the C-terminal region of MvaL10 shows exceptionally high similarity to the C-terminal sequence of the MvaL12 protein. This is not the case for the E. coli proteins but was also observed for the human, Halobacterium and Sulfolobus proteins.

Amino Acid Sequence↗

PubMed1989

The complete amino acid sequence of ribosomal protein S18 from the moderate thermophile Bacillus stearothermophilus.

The amino acid sequence of ribosomal protein S18 from Bacillus stearothermophilus has been completely determined by automated sequence analysis of the intact protein as well as of peptides derived from digestion with Staphylococcus aureus protease at pH 4.0 and cleavage with cyanogen bromide. The carboxy-terminal region was verified by both amino acid analyses of chymotryptic peptides and by mass spectrometry from the terminal region. The protein contains 77 amino acid residues and has an Mr of 8838. Comparison of this sequence with the sequences of the S18 proteins from tobacco and liverwort chloroplasts and E. coli shows a relatively high similarity, ranging from 42 to 55% identical residues with the B. stearothermophilus S18 protein. The regions of homology common to all four proteins consist of several positively charged sections spanning the entire length of the protein.

Amino Acid Sequence↗

PubMed1989

Complete nucleotide sequence and gene organization of the broad-host-range plasmid RSF1010.

We present the complete nucleotide sequence of RSF1010, a naturally occurring broad-host-range plasmid belonging to the Escherichia coli incompatibility group Q and encoding resistance to streptomycin and sulfonamides. A molecule of RSF1010 DNA consists of 8684 bp and has a G + C content of 61%. Analysis of the distribution of translation start and stop codons in the sequence has revealed the existence of more than 40 open reading frames potentially capable of encoding polypeptides of 60 or more amino acids. To date, products of eleven such potential RSF1010 genes have been identified through the application of controlled expression vector systems, and for eight of them, the reading frame has been confirmed by N- and/or C-terminal amino acid sequence determinations on the purified proteins. The sequencing results are discussed in relation to the systems of replication, host range, conjugal mobilization and antibiotic resistance determinants associated with the RSF1010 plasmid.

Amino Acid Sequence↗

PubMed1989

Cyclophilin and peptidyl-prolyl cis-trans isomerase are probably identical proteins.

The enzyme peptidyl-prolyl cis-trans isomerase (PPIase) was recently discovered in mammalian tissues and purified from porcine kidney. It catalyses the slow cis-trans isomerization of proline peptide (Xaa-Pro) bonds in oligopeptides and accelerates slow, rate-limiting steps in the folding of several proteins. Here, we report the N-terminal sequence of PPIase together with further chemical and enzymatic properties. The results indicate that this enzyme is probably identical to cyclophilin, a recently discovered mammalian protein which binds tightly to cyclosporin A (CsA). Cyclophilin is thought to be linked to the immunosuppressive action of CsA. The first 38 amino-acid residues of porcine PPIase and of bovine cyclophilin are identical and the two proteins both have a relative molecular mass of about 17,000 (ref. 7). The catalysis of prolyl isomerization in oligopeptides and of protein folding by PPIase are strongly inhibited in the presence of low levels of CsA. The activities of both PPIase and cyclophilin depend on a single sulphydryl group. At present it is unknown whether the inhibition of prolyl isomerase activity is related with the immunosuppressive action of CsA.

Amino Acid Isomerases↗

PubMed1989

Topography of surface-exposed amino acids in the membrane protein bacteriorhodopsin determined by proteolysis and micro-sequencing.

The topography of membrane-surface-exposed amino acids in the light-driven proton pump bacteriorhodopsin (BR) was studied. By limited proteolysis of purple membrane with papain or proteinase K, domains were cleaved, separated by SDS-PAGE, and electroblotted onto polyvinylidene difluoride (PVDF) membranes. Fragments transferred were sequenced in a gas-phase sequencer. Papain cleavage sites at Gly-65, Gly-72, and Gly-231, previously only deduced from the apparent molecular weight of the digestion fragments, could be confirmed by N-terminal micro-sequencing. By proteinase K, cleavage occurred at Gln-3, Phe-71, Gly-72, Tyr-131, Tyr-133, and Ser-226, i.e., in regions previously suggested to be surface-exposed. Additionally, proteinase-K cleavage sites at Thr-121 and Leu-127 were identified, which are sites predicted to be in the alpha-helical membrane-spanning segment D. Our results, especially that the amino acids Gly-122 to Tyr-133 are protruding into the aqueous environment, place new constraints on the amino-acid folding of BR across the purple membrane. The validity of theoretical prediction methods of the secondary structure and polypeptide folding for membrane proteins is challenged. The results on BR show that micro-sequencing of peptides separated by SDS-PAGE and blotted to PVDF can be successfully applied to the study of membrane proteins.

Amino Acid Sequence↗