Direct Measurements of the Cobalt-Thiolate Bonds Strength in Rubredoxin by Single-Molecule Force Spectroscopy

Cobalt is a trace transition metal. Although it is not abundant on earth, tens of cobalt-containing proteins exist in life. Moreover, the characteristic spectrum of Co(II) ion makes it a powerful probe for the characterization of metal-binding proteins through the formation of cobalt-ligand bonds. Since most of these natural and artificial cobalt-containing proteins are stable, we believe that these cobalt-ligand bonds in the protein system are also mechanically stable. To prove this, we used atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) to directly measure the rupture force of Co(II)-thiolate bond in Co-substituted rubredoxin (CoRD). By combining the chemical denature/renature method for building metalloprotein and cysteine coupling-based polyprotein construction strategy, we successfully prepared the polyprotein sample (CoRD)(n) suitable for single-molecule studies. Thus, we quantified the strength of Co(II)-thiolate bonds in rubredoxin with a rupture force of similar to 140 pN, revealing that it is a mechanostable chemical bond. In addition, the Co-S bond is more labile than the Zn-S bond in proteins, similar to the result from the metal-competing titration experiment.

Automated summary

Cobalt is a trace transition metal that exists in numerous cobalt-containing proteins in living organisms. The Co(II) ion can be used as a powerful tool to characterize metal-binding proteins through the formation of cobalt-ligand bonds. This study used atomic force microscopy-based single-molecule force spectroscopy to directly measure the rupture force of the Co(II)-thiolate bond in cobalt-substituted rubredoxin. The results showed that the Co(II)-thiolate bond is a mechanically stable chemical bond, and the Co-S bond is less stable compared to the Zn-S bond in proteins.