Single molecule force spectroscopy reveals that the oxidation state of cobalt ions plays an important role in enhancing the mechanical stability of proteins

Engineered bi-histidine (biHis)-based metal chelation is a general and robust method to enhance the mechanical stability of proteins. Here we used single molecule force spectroscopy techniques to investigate the effect of binding of Co²⁺/Co³⁺ on the mechanical stability of an engineered biHis mutant of protein GB1, G6-53. We found that the binding of Co²⁺/Co³⁺ can lead to an enhancement of the mechanical stability of G6-53, but the degree of enhancement is drastically different. The binding of Co²⁺ can only lead to marginal enhancement of G6-53's mechanical stability, while Co³⁺ has a much stronger effect. This large difference is likely due to the large difference in thermodynamic stability and kinetic lability of Co²⁺ and Co³⁺ complexes. These results opened up new avenues towards fine tuning the mechanical properties of proteins.