title: Cooperative binding of PhoB(DBD) to its cognate DNA sequence-a combined application of single-molecule and ensemble methods.
abstract: A combined approach based on isothermal titration calorimetry (ITC), fluorescence resonance energy transfer (FRET) experiments, circular dichroism spectroscopy (CD), atomic force microscopy (AFM) dynamic force spectroscopy (DFS), and surface plasmon resonance (SPR) was applied to elucidate the mechanism of protein-DNA complex formation and the impact of protein dimerization of the DNA-binding domain of PhoB (PhoB(DBD)). These insights can be translated to related members of the family of winged helix-turn-helix proteins. One central question was the assembly of the trimeric complex formed by two molecules of PhoB(DBD) and two cognate binding sites of a single oligonucleotide. In addition to the native protein WT-PhoB(DBD), semisynthetic covalently linked dimers with different linker lengths were studied. The ITC, SPR, FRET, and CD results indicate a positive cooperative binding mechanism and a decisive contribution of dimerization on the complex stability. Furthermore, an alanine scan was performed and binding of the corresponding point mutants was analyzed by both techniques to discriminate between different binding types involved in the protein-DNA interaction and to compare the information content of the two methods DFS and SPR. In light of the published crystal structure, four types of contribution to the recognition process of the pho box by the protein PhoB(DBD) could be differentiated and quantified. Consequently, it could be shown that investigating the interactions between DNA and proteins with complementary techniques is necessary to fully understand the corresponding recognition process.
authors.affiliation: Organic and Bioorganic Chemistry and ‡Experimental Biophysics, Bielefeld University , Universitaetsstrasse 25, 33615 Bielefeld, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Ritzefeld
authors.forename: Volker
authors.initials: V
authors.lastname: Walhorn
authors.forename: Christin
authors.initials: C
authors.lastname: Kleineberg
authors.forename: Adeline
authors.initials: A
authors.lastname: Bieker
authors.forename: Klaus
authors.initials: K
authors.lastname: Kock
authors.forename: Christian
authors.initials: C
authors.lastname: Herrmann
authors.forename: Dario
authors.initials: D
authors.lastname: Anselmetti
authors.forename: Norbert
authors.initials: N
authors.lastname: Sewald
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D001483:Base Sequence; D002942:Circular Dichroism; D004269:DNA, Bacterial / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D048168:Escherichia coli K12; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D019077:Helix-Turn-Helix Motifs; D018625:Microscopy, Atomic Force; D017354:Point Mutation; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C585752:PhoB protein, E coli; D014157:Transcription Factors; C061278:PhoB protein, Bacteria