Zinc fingers are highly ubiquitous structural motifs that provide stability to proteins, thus contributing to their correct folding. Despite the high thermodynamic stability of the ZnCys4 centers, their kinetic properties display remarkable lability. Here, we use a combination of protein engineering with single molecule force spectroscopy atomic force microscopy (AFM) to uncover the surprising mechanical lability (∼90 pN) of the individual Zn-S bonds that form the two equivalent zinc finger motifs embedded in the structure of the multidomain DnaJ chaperone. Rational mutations within the zinc coordinating residues enable direct identification of the chemical determinants that regulate the interplay between zinc binding-requiring the presence of all four cysteines-and disulfide bond formation. Finally, our observations show that binding to hydrophobic short peptides drastically increases the mechanical stability of DnaJ. Altogether, our experimental approach offers a detailed, atomistic vista on the fine chemical mechanisms that govern the nanomechanics of individual, naturally occurring zinc finger.
Keywords: atomic force microscopy; biophysics; force spectroscopy; metalloproteins; nanomechanics; single molecule.