title: Single-molecule experiments to elucidate the minimal requirement for DNA recognition by transcription factor epitopes.
abstract: Interactions between proteins and DNA are essential for the regulation of cellular processes in all living organisms. In this context, it is of special interest to investigate the sequence-specific molecular recognition between transcription factors and their cognate DNA sequences. As a model system, peptide and protein epitopes of the DNA-binding domain (DBD) of the transcription factor PhoB from Escherichia coli are analyzed with respect to DNA binding at the single-molecule level. Peptides representing the amphiphilic recognition helix of the PhoB DBD (amino acids 190-209) are chemically synthesized and C-terminally modified with a linker for atomic force microscopy-dynamic force spectroscopy experiments (AFM-DFS). For comparison, the entire PhoB DBD is overexpressed in E. coli and purified using an intein-mediated protein purification method. To facilitate immobilization for AFM-DFS experiments, an additional cysteine residue is ligated to the protein. Quantitative AFM-DFS analysis proves the specificity of the interaction and yields force-related properties and kinetic data, such as thermal dissociation rate constants. An alanine scan for strategic residues in both peptide and protein sequences is performed to reveal the contributions of single amino acid residues to the molecular-recognition process. Additionally, DNA binding is substantiated by electrophoretic mobility-shift experiments. Structural differences of the peptides, proteins, and DNA upon complex formation are analyzed by circular dichroism spectroscopy. This combination of techniques eventually provides a concise picture of the contribution of epitopes or single amino acids in PhoB to DNA binding.
authors.affiliation: Organic and Bioorganic Chemistry, Department of Chemistry, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Wollschläger
authors.forename: Katharina
authors.initials: K
authors.lastname: Gaus
authors.forename: André
authors.initials: A
authors.lastname: Körnig
authors.forename: Rainer
authors.initials: R
authors.lastname: Eckel
authors.forename: Sven-David
authors.initials: SD
authors.lastname: Wilking
authors.forename: Matthew
authors.initials: M
authors.lastname: McIntosh
authors.forename: Zsuzsanna
authors.initials: Z
authors.lastname: Majer
authors.forename: Anke
authors.initials: A
authors.lastname: Becker
authors.forename: Robert
authors.initials: R
authors.lastname: Ros
authors.forename: Dario
authors.initials: D
authors.lastname: Anselmetti
authors.forename: Norbert
authors.initials: N
authors.lastname: Sewald
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000378:metabolism*; D018625:Microscopy, Atomic Force / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D013057:Spectrum Analysis / Q000379:methods; D014157:Transcription Factors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D000939:Epitopes; D014157:Transcription Factors; D004247:DNA