Gene expression in eukaryotes is controlled at the transcriptional level by the specific binding of transcription factors to defined DNA sequences. In this way, cell growth, differentiation, and development are regulated. The possibility to influence and control cell metabolism through modified synthetic transcription factors [1][2][3][4] offers fascinating prospects for molecular cell biology in the framework of biomimetics and synthetic biology. [5,6] The design and synthesis of biologically active artificial enzymes and new protein-based materials can be investigated by the combination of bioorganic bottom-up synthesis and single-molecule affinity nanotechnology. With this approach important questions can be addressed, such as the extent to which a single recognition helix contributes to the specific binding of a complete protein to DNA, the effect that a single amino acid point mutation has upon biological specificity and affinity, and the minimal peptide sequence length to ensure binding specificity. This approach would also aid in the design of artificial proteins that contain a purely synthetic helix-turn-helix (HTH) binding motif.In this context, it is of considerable interest to elucidate the DNA-binding specificity of synthetic peptides with a primary sequence akin to the binding domain of a transcription factor. We studied a 20-residue peptide that represents the native sequence of a binding epitope of the transcription activator PhoB (E. coli) and three single point mutants of this peptide.PhoB is a transcription activator which, after phosphorylation by PhoR, binds to the phosphate box in the promoter region of the phosphate regulon pho and activates the expression of genes involved in phosphate metabolism.