DNA-protein interactions are a key element in the regulation of cellular processes. Therefore, it is of special interest to investigate sequence-specific molecular recognition between transcription factors and their corresponding DNA sequences.
DNA binding of peptide and protein epitopes of the DNA-binding domain (DBD) of the transcription factor PhoB from E. coli was analyzed as a model system. Structurally, PhoB belongs to the family of winged helix-turn-helix proteins and controls the expression of genes involved in phosphate metabolism. It is composed of a transactivation domain and the DBD that binds to specific DNA sequences. Peptide design was assisted by structural information, derived from X-ray single crystal and NMR solution structures of the DBD both free and in complex with DNA.
C-terminally modified peptide epitopes representing the amphiphilic DNA recognition helix were chemically synthesized using microwave assisted solid phase peptide synthesis. For comparison, the entire PhoB DBD was overexpressed in E. coli, purified using intein-mediated protein purification and further modified by native chemical ligation.
Molecular recognition and DNA binding was investigated on the single molecule level. Quantitative atomic force spectroscopy analysis proved the specificity of the interaction and yielded force-related properties and kinetic data, such as thermal dissociation rate constants. An alanine scan for strategic residues in both the peptide and protein sequences was performed to reveal the contributions of single amino acid residues to the molecular recognition process. Sequence specific DNA-binding of both peptide and protein was additionally proven in competition experiments.
DNA binding was also substantiated by ensemble methods such as electrophoretic mobility shift experiments and surface plasmon resonance in order to correlate single molecule data with macroscopic data. Structural properties of the peptides, proteins, DNA-protein complexes were analyzed by circular dichroism spectroscopy. This combination of techniques provides a concise picture of the contribution of epitopes or single amino acids in PhoB to DNA binding.
This project is supported by DFG (SFB 613).
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