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M Bantscheff

Publications and source records attributed to M Bantscheff.

5 recordsLinked to original sources
PubMed2001

Rational design and molecular characterization of a chimaeric response regulator protein.

BvgA and EvgA are closely related response regulators from Bordetella pertussis and Escherichia coli. To analyze the domain borders and linker sequences of these proteins, we used limited proteolysis and matrix-assisted laser desorption/ionization-mass spectrometry analysis of the in-gel-digested proteolytic fragments. The thermolysin-sensitive linker regions were found to extend from Leu130 to Thr144 for BvgA and from Leu127 to Ser133 for EvgA. These data provided the rationale for the construction of the chimaeric protein HA. HA carries the EvgA receiver and BvgA output domains, fused in the central part of the linker sequences of the parent proteins. Thermolysin-sensitive sites of HA were found at positions identical with those in the EvgA and BvgA linker sequences, indicating intact folding of its receiver and output domains. Consistent with this, the chimaera showed virtually unchanged phosphorylation and dimerization properties. However, BvgA and HA differed in the effect of phosphorylation on their DNA-binding activities. In the case of BvgA, phosphorylation resulted in an increased affinity and specificity in DNA binding, whereas the DNA-binding properties of HA were not affected by phosphorylation. The chimaera HA was unable to activate transcription of the BvgA-dependent fha promoter, either in vivo or in vitro. These results indicate that the phosphorylation-induced activation of BvgA requires specific interactions between the receiver and output domains that are disturbed in the chimaera.

Amino Acid Sequence↗
PubMed2000

Dimerization of signalling modules of the EvgAS and BvgAS phosphorelay systems.

Biophysical and biochemical properties of signalling proteins or domains derived from the unorthodox EvgAS and BvgAS two-component phosphorelay systems of Escherichia coli and Bordetella pertussis were investigated. Oligomerization of the effector proteins EvgA and BvgA and of truncated EvgS and BvgS derived signalling proteins containing the receiver and histidine containing phosphotransfer (HPt) domains or comprising only the HPt domains were characterized by native gel electrophoresis, gel permeation experiments and analytical ultracentrifugation. The results obtained by the different methods are consistent with non-phosphorylated EvgA and BvgA proteins being dimers in solution with a dissociation constant significantly below 1 microM. In contrast, all sensor derived domains of EvgS and BvgS were observed to be monomers in vitro. No indications for a phosphorylation induced stimulation of oligomerization of the C-terminal histidine kinase domains could be detected. In agreement with these data, surface plasmon resonance studies revealed a 2:1 stoichiometry in the interaction of EvgA with the immobilized EvgS HPt domain and an affinity constant of 1. 24x10(6) M(-1).

Bordetella pertussis↗
PubMed2000

Structure-function relationships in the Bvg and Evg two-component phosphorelay systems.

The unorthodox two-component phosphorelay systems BvgAS and EvgAS of Bordetella pertussis and E. coli, respectively, are suitable model systems to investigate the molecular basis of signalling specificity, because, despite their high relatedness on the sequence level, they do not cross-talk to each other. We could show that the two systems belong to the obligate type of phosphorelay systems and that signalling specificity is mediated by the HPt modules of the histidine kinases and the receiver domains of the effector proteins. To gain more insight into signalling specificity on the molecular level, we started a detailed structural analysis of the respective proteins using a combination of genetic and biochemical methods including limited proteolysis and chemical modification of purified proteins and their mass spectrometrical analysis.

Amino Acid Sequence↗
PubMed1999

Identification of linker regions and domain borders of the transcription activator protein NtrC from Escherichia coli by limited proteolysis, in-gel digestion, and mass spectrometry.

We have developed a mass spectrometry based method for the identification of linker regions and domain borders in multidomain proteins. This approach combines limited proteolysis and in-gel proteolytic digestions and was applied to the determination of linkers in the transcription factor NtrC from Escherichia coli. Limited proteolysis of NtrC with thermolysin and papain revealed that initial digestion yielded two major bands in SDS-PAGE that were identified by mass spectrometry as the R-domain and the still covalently linked OC-domains. Subsequent steps in limited proteolysis afforded further cleavage of the OC-fragment into the O- and the C-domain at accessible amino acid residues. Mass spectrometric identification of the tryptic/thermolytic peptides obtained after in-gel total proteolysis of the SDS-PAGE-separated domains determined the domain borders and showed that the protease accessible linker between R- and O-domain comprised amino acids Val-131 and Gln-132 within the "Q-linker" in agreement with papain and subtilisin digestion. The region between amino acid residues Thr-389 and Gln-396 marked the hitherto unknown linker sequence that connects the O- with the C-domain. High abundances of proline-, alanine-, serine-, and glutamic acid residues were found in this linker structure (PASE-linker) of related NtrC response regulator proteins. While R- and C-domains remained stable under the applied limited proteolysis conditions, the O-domain was further truncated yielding a core fragment that comprised the sequence from Ile-140 to Arg-320. ATPase activity was lost after separation of the R-domain from the OC-fragment. However, binding of OC- and C- fragments to specific DNA was observed by characteristic band-shifts in migration retardation assays, indicating intact tertiary structures of the C-domain. The outlined strategy proved to be highly efficient and afforded lead information of tertiary structural features necessary for protein design and engineering and for structure-function studies.

Amino Acid Sequence↗