A novel class of molecular chaperones co-ordinates the assembly and targeting of complex metalloproteins by binding to an amino-terminal peptide of the cognate substrate. We have previously shown that the NarJ chaperone interacts with the N-terminus of the NarG subunit coming from the nitrate reductase complex, NarGHI. In the present study, NMR structural analysis revealed that the NarG(1-15) peptide adopts an alpha-helical conformation in solution. Moreover, NarJ recognizes and binds the helical NarG(1-15) peptide mostly via hydrophobic interactions as deduced from isothermal titration calorimetry analysis. NMR and differential scanning calorimetry analysis revealed a modification of NarJ conformation during complex formation with the NarG(1-15) peptide. Isothermal titration calorimetry and BIAcore experiments support a model whereby the protonated state of the chaperone controls the time dependence of peptide interaction.