Single-molecule DNA force spectroscopy to probe interactions with the tri-peptide Lys-Trp-Lys.

Interactions between nucleotides and proteins are essential in the life cycle of cells. Specific physico-chemical properties of the system give rise to diverse modes of interaction. Coulomb interactions between charged residues on either macromolecule provide an important contribution to the interaction potential. For uncharged residues, the interaction potential is dominated by van der Waals interactions. A special form of van der Waals interactions is intercalation, which can be understood as a stacking interaction between hydrophobic aromatic residues to avoid exposure to polar solvents. A well-known example of stacking is provided by DNA. A helical motif stacks the base molecules and stabilizes the structure by base–base interactions and shielding from the solvent. The aromatic amino acids in proteins have also been noted as structural elements with an ability to intercalate. In the helix-destabilizing DNA-binding proteins gp32 of bacteriophage T4 and gp5 of bacteriophage M13, repeats of tyrosine and/or tryptophan residues have been suggested to exemplify the importance of intercalation in DNA–protein interactions. Recent studies with optical tweezers have shown that single-molecule DNA experiments in the presence of intercalating molecules enable a study of the “molecular force” exerted while molecules intercalate. Structural parameters of the DNA influenced by the interaction can be directly obtained, namely changes in contour and persistence lengths, binding constant and binding site size as a function of applied force. The binding affinity of intercalating dye molecules (ethidium bromide, YO-1 and YOYO-1) is usually quite large, while the binding affinity of model-peptide systems containing an intercalating aromatic amino acid tryptophan or tyrosine is much smaller. Based on our work on DNA–dye molecule interactions, we investigate herein the interaction of single-molecule DNA with the tripeptide LysTrp-Lys using optical tweezers force spectroscopy. In pioneering work by H l ne and Brun using fluorescence spectroscopy, a two-step binding mechanism [Eq. (1)] based on the observed increase in binding constant as a function of nucleotide concentration has been proposed [Eq. (1)]:

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