Abstract
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.
Highlights
The pioneeringfolding study of Ribonuclease A by Nobel laureate Anfinsen takes advantage of reductive denaturation of the stable S-S bonds inside the protein and provides the first picture of how protein folds and unfolds[9]
A worm-like chain (WLC) model which describes the function between applied force and polymer extension, multiple unfolding steps (>2) with the combination ΔLc of ~9 nm can be detected, besides GB1 unfolding events (Fig. 2b)
Our results indicate that we observed multistep protein unfolding scenarios from the rupture of the
Summary
The pioneering (un)folding study of Ribonuclease A by Nobel laureate Anfinsen takes advantage of reductive denaturation of the stable S-S bonds inside the protein and provides the first picture of how protein folds and unfolds[9]. The protein unfolding provides an unambiguous marker to identify single disulfide bond reduction event and many unique S-S bond properties are revealed Inspired by these works, we chose metalloprotein with relatively stable and multiple metal-ligand coordination bonds, to demonstrate the multistep unfolding phenomena of protein. It naturally eliminates the most contribution of non-covalent interactions for protein structure and stability These Cd-S bonds determine the 3D conformation of MT, and the rupture scenario of the metal clusters equals the MT unfolding. It has been applied to the study of metal-ligand coordination bond strength, chemical reactivity and the interplay between the metal center and the protein structure in metalloprotein systems[50,51,52,53] We chose it to detect multistep rupture scenarios of Cd3S9, which corresponds to metallothionein unfolding
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