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Force-induced topological changes of proteins studied at the single-molecule level He, Chengzhi

Abstract

The knotted polypeptide chain is one of the most surprising topological features found in certain proteins. Understanding how knotted proteins overcome the topological difficulty during the folding process has become a challenging problem. Theoretical studies suggest that a slipknotted structure can serve as an important intermediate in the transformation from unknotted structure to a knotted structure. This thesis mainly focuses on the mechanical folding/unfolding of a slipknotted protein, the ORF109 of the Acidianus Filamentous Virus 3 (AFV3-109), using single-molecule force spectroscopy (SMFS). To show the power of atomic force microscopy (AFM) in the study of SMFS, an α/β protein, NuG2, is stretched and relaxed at a single-molecule level using AFM and the mechanical unfolding and folding events are directly observed. By applying force onto AFV3-109 in different directions, we are able to untie the slipknot to a linear polypeptide chain, as well as tighten it into a trefoil knot involving ∼ 13 amino acid residues. Multiple pathways of untying and tightening are found by both SMFS experiments and Steered Molecular Dynamics (SMD) simulations, revealing that the kinetic partitioning mechanism governs the unfolding of the slipknotted protein. In addition, SMD simulations provide detailed molecular mechanisms of the unfolding of the protein and the topological changes from a slipknot to a linear chain, as well as from a slipknot to a trefoil knot. Moreover, the mechanical folding of AFV3-109 is directly observed using optical tweezers, providing new insight into the folding mechanism of knotted/slipknotted proteins.

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Attribution-NonCommercial-NoDerivs 2.5 Canada

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