Ligand modulated parallel mechanical unfolding pathways of Maltose binding proteins (MBPs)

Protein folding/unfolding are complex phenomena and it is accepted that multidomain proteins generally follow multiple pathways. Maltose binding protein is a large (a two-domain 370 amino acid residue) bacterial periplasmic protein involved in maltose uptake. Despite the large size, it has been shown to exhibit an apparent two-state equilibrium unfolding in bulk experiments. Single-molecule studies can uncover rare events that are masked by averaging in bulk studies. Here, we use single-molecule force spectroscopy to study the mechanical unfolding pathways of maltose binding protein (MBP) and its precursor protein (preMBP) in the presence and absence of ligands. Our results show that MBP exhibits kinetic partitioning on mechanical stretching and unfolds via two parallel pathways: one of them involves a mechanically stable intermediate (path I) while the other is devoid of it (path II). The apoMBP unfolds via path I in 62% of the mechanical unfolding events and the remaining 38% follow path II. In the case of maltose bound MBP, the protein unfolds via the intermediate in 79% of the cases, the remaining 21% via path II. Similarly, on binding to maltotriose, a ligand whose binding strength with the polyprotein is similar to that of maltose, the occurrence of the intermediate is comparable (82% via path I) to that of maltose. The precursor protein preMBP also shows a similar behaviour upon mechanical unfolding. The percentages of molecules unfolding via path I are 53% in the apo form and 68% and 72% upon binding to maltose and maltotriose, respectively for preMBP