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In case of an oligonucleotide containing only an AT-rich minor groove in position 5’ and aTGTCA-motif in position 3’ no binding was observed (Table 1, entry 4). This resultindicates that both binding sites (the TGTCA motif and the AT-rich minor groove) areessential for the binding process.Table 2. AFM dynamic force spectroscopy of PhoB peptides and proteins using pho boxDNA [2,3]. k off =dissociation rate constant for peptide/protein DNA complexes. τ=1/k offNamek off [s -1 ]peptidePhoB(190-209)τ [s]k off [s -1 ]proteinPhoB(127-229)WT(wildtype)3.1 ± 2.1 0.32 1.4 400R193A 0.071 ± 0.053 14 14.5 83H198A 49.5 ± 21.2 0.020 21.9 1R203A --- --- --- ---Single molecule force spectroscopy experiments were performed to determine the kineticoff-rates of peptide and protein epitopes (Table 1). DNA molecules were covalently boundto the AFM tip and the PhoB epitopes to the surface. To elucidate the binding contributionsof certain amino acids, the corresponding residues were substituted by alanine [2,3]. Allresults are comparable for the protein and the peptide mutants. DNA-protein/peptidebinding could be observed for R193A and H198A whereas mutant R203A did not bind toDNA (Table 2). Taking into consideration that the arginine at position 203 is conservedwithin the PhoB family, these results emphasize the relevance of this residues for DNAbinding [2,3].τ [s]Fig. 2. AFM competition experiments using PhoB(127-229)WT, Pho box DNA and PhoB(190-209)WT as competitor [2,3].To prove the sequence specific DNA-binding of both, peptides and proteins, competitionexperiments were accomplished (Figure 2) [2,3]. Therefore force spectroscopy experimentsof PhoB(127-229)WT with pho box DNA were performed. Addition of the competitorPhoB(190-209)WT resulted in an overall decrease of binding. The washing step couldrestore binding. These results implicate that the recognition process is specific in both cases[2,3].AcknowledgmentsThis work was supported by the DFG (SFB 613) and the German National Academic Foundation(Studienstiftung des Deutschen Volkes (PhD scholarship to Markus Ritzefeld)).References1. Makino, K., et al. Mol. Biol. 259, 15-26 (1996).2. Wollschläger, K., et al. Small 5, 484-495 (2008).3. Eckel, R., et al. Angew. Chem. 117, 3989-2933 (2005); Angew. Chem. Int. Ed. 44, 3921-3924(2005).479