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UBC Theses and Dissertations

Investigating force-induced unfolding and folding of proteins at the single molecule level Wang, Han


Understanding the molecular mechanism of protein folding has always remained a challenging problem. Extensive investigations based on ensemble methods have been carried out to shed light on the protein folding problem. Compared with ensemble-averaged measurements, single-molecule force spectroscopy (SMFS) has evolved as a powerful technique to probe mechanical unfolding/folding of proteins at the single-molecule level, which provides rich details of protein conformational changes. Optical tweezers (OT), as one representative SMFS technique, have been widely used to directly monitor a protein folding process. This thesis presents a series of studies that use OT to investigate the mechanical unfolding/folding of two distinct classes of proteins: knotted/slipknotted proteins and Ca²⁺-binding metalloproteins. First, we combined OT and steered molecular dynamics (SMD) simulations to investigate a complex slipknotted protein. When stretched from the N- and C-termini, the slipknotted structure could be completely untied via multiple unfolding pathways. Upon relaxation, this protein showed complex folding behaviors involving misfolding. Our studies demonstrate the unfolding/folding mechanisms of a slipknotted protein with a complex topological conformation. Second, we used OT to mechanically tighten, untie and retie a trefoil knotted protein. Our results revealed that protein folding with a preformed knot was fast and robust; however, the folding from the unfolded polypeptide chain without the knot conformation was significantly slower, suggesting that the knotting was the rate-limiting step for the folding of this trefoil knotted protein. Next, we investigated the mechanical unfolding/folding behaviors of a Ca²⁺-binding protein, RTX (repeats-in-toxin) block V. Our results elucidated the secretion mechanism of this RTX block V and revealed that the mechanical design can ensure an efficient translocation process. Finally, we used OT to study another Ca²⁺-binding protein, RTX block IV. The mechanical properties of RTX block IV was well-characterized. Our results revealed that RTX block IV follows a similar translocation mechanism to be secreted as RTX block V. We also suggested that the secreted RTX block V is stabilized by folded RTX block IV and protected from being unfolded in vivo. Overall, this thesis clarified the unfolding/folding mechanisms of two knotted/slipknotted proteins and Ca²⁺-binding protein RTX.

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