Transforming protein αD into a mechanically stable protein by zinc binding.

αD is a designed three-helix bundle protein. Like most naturally occurring helical proteins, it is mechanically labile with an unfolding force of <15 pN, revealed by atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). This protein has been further designed with a tri-cysteine metal-binding site, named αDIV, which can bind heavy transition metals. Here, we demonstrate that incorporating such a metal-binding site can transform this mechanically labile protein into a stable one. We show that zinc binds to the tri-cysteine site and increases the unfolding force to ∼160 pN. This force is one order of magnitude higher than that of the apo-protein (<15 pN). Moreover, the unfolding mechanism of Zn-αDIV indicates the correct zinc binding with the tri-cysteine site, forming three mechanostable Zn-thiolate bonds. Thus, αDIV could be a potential α-helical structure-based building block for synthesizing biomaterials with tunable mechanical properties.