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 ∼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.
Keywords: biophysics; cobalt-thiolate bonds; metalloproteins; rubredoxin; single molecule-force spectroscopy.
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