title: Mechanically untying a protein slipknot: multiple pathways revealed by force spectroscopy and steered molecular dynamics simulations.
abstract: Protein structure is highly diverse when considering a wide range of protein types, helping to give rise to the multitude of functions that proteins perform. In particular, certain proteins are known to adopt a knotted or slipknotted fold. How such proteins undergo mechanical unfolding was investigated utilizing a combination of single molecule atomic force microscopy (AFM), protein engineering, and steered molecular dynamics (SMD) simulations to show the mechanical unfolding mechanism of the slipknotted protein AFV3-109. Our results reveal that the mechanical unfolding of AFV3-109 can proceed via multiple parallel unfolding pathways that all cause the protein slipknot to untie and the polypeptide chain to completely extend. These distinct unfolding pathways proceed via either a two- or three-state unfolding process involving the formation of a well-defined, stable intermediate state. SMD simulations predict the same contour length increments for different unfolding pathways as single molecule AFM results, thus providing a plausible molecular mechanism for the mechanical unfolding of AFV3-109. These SMD simulations also reveal that two-state unfolding is initiated from both the N- and C-termini, while three-state unfolding is initiated only from the C-terminus. In both pathways, the protein slipknot was untied during unfolding, and no tightened slipknot conformation was observed. Detailed analysis revealed that interactions between key structural elements lock the knotting loop in place, preventing it from shrinking and the formation of a tightened slipknot conformation. Our results demonstrate the bifurcation of the mechanical unfolding pathway of AFV3-109 and point to the generality of a kinetic partitioning mechanism for protein folding/unfolding.
authors.affiliation: Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1 Canada.
authors.forename: Chengzhi
authors.initials: C
authors.lastname: He
authors.forename: Georgi Z
authors.initials: GZ
authors.lastname: Genchev
authors.forename: Hui
authors.initials: H
authors.lastname: Lu
authors.forename: Hongbin
authors.initials: H
authors.lastname: Li
mesh_terms: D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*
chemical_list: D011506:Proteins