Multistep Protein Unfolding Scenarios from the Rupture of a Complex Metal Cluster Cd₃S₉

Protein (un)folding is a complex and essential process. With the rapid development of single-molecule techniques, we can detect multiple and transient proteins (un)folding pathways/intermediates. However, the observation of multiple multistep (>2) unfolding scenarios for a single protein domain remains limited. Here, we chose metalloprotein with relatively stable and multiple metal-ligand coordination bonds as a system for such a purpose. Using AFM-based single-molecule force spectroscopy (SMFS), we successfully demonstrated the complex and multistep protein unfolding scenarios of the β-domain of a human protein metallothionein-3 (MT). MT is a protein of ~60 amino acids (aa) in length with 20 cysteines for various metal binding, and the β-domain (βMT) is of ~30 aa with an M₃S₉ metal cluster. We detected four different types of three-step protein unfolding scenarios from the Cd-βMT, which can be possibly explained by the rupture of Cd-S bonds in the complex Cd₃S₉ metal cluster. In addition, complex unfolding scenarios with four rupture peaks were observed. The Cd-S bonds ruptured in both single bond and multiple bonds modes. Our results provide not only evidence for multistep protein unfolding phenomena but also reveal unique properties of metalloprotein system using single-molecule AFM.