title: Knotting and unknotting of a protein in single molecule experiments.
abstract: Spontaneous folding of a polypeptide chain into a knotted structure remains one of the most puzzling and fascinating features of protein folding. The folding of knotted proteins is on the timescale of minutes and thus hard to reproduce with atomistic simulations that have been able to reproduce features of ultrafast folding in great detail. Furthermore, it is generally not possible to control the topology of the unfolded state. Single-molecule force spectroscopy is an ideal tool for overcoming this problem: by variation of pulling directions, we controlled the knotting topology of the unfolded state of the 52-knotted protein ubiquitin C-terminal hydrolase isoenzyme L1 (UCH-L1) and have therefore been able to quantify the influence of knotting on its folding rate. Here, we provide direct evidence that a threading event associated with formation of either a 31 or 52 knot, or a step closely associated with it, significantly slows down the folding of UCH-L1. The results of the optical tweezers experiments highlight the complex nature of the folding pathway, many additional intermediate structures being detected that cannot be resolved by intrinsic fluorescence. Mechanical stretching of knotted proteins is also of importance for understanding the possible implications of knots in proteins for cellular degradation. Compared with a simple 31 knot, we measure a significantly larger size for the 52 knot in the unfolded state that can be further tightened with higher forces. Our results highlight the potential difficulties in degrading a 52 knot compared with a 31 knot.
authors.affiliation: Physik Department E22, Technische Universität München, 85748 Garching, Germany;
authors.forename: Fabian
authors.initials: F
authors.lastname: Ziegler
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom; Faculty of Sciences, Universiti Brunei Darussalam, Gadong BE 1410, Brunei Darussalam;
authors.forename: Nicole C H
authors.initials: NC
authors.lastname: Lim
authors.affiliation: Physik Department E22, Technische Universität München, 85748 Garching, Germany;
authors.forename: Soumit Sankar
authors.initials: SS
authors.lastname: Mandal
authors.affiliation: Physik Department E22, Technische Universität München, 85748 Garching, Germany;
authors.forename: Benjamin
authors.initials: B
authors.lastname: Pelz
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom;
authors.forename: Wei-Ping
authors.initials: WP
authors.lastname: Ng
authors.affiliation: B CUBE, Center for Molecular Bioengineering, Technische Universität Dresden, 01307 Dresden, Germany;
authors.forename: Michael
authors.initials: M
authors.lastname: Schlierf
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom; sej13@cam.ac.uk mrief@ph.tum.de.
authors.forename: Sophie E
authors.initials: SE
authors.lastname: Jackson
authors.affiliation: Physik Department E22, Technische Universität München, 85748 Garching, Germany; Munich Center for Integrated Protein Science, 81377 Munich, Germany sej13@cam.ac.uk mrief@ph.tum.de.
authors.forename: Matthias
authors.initials: M
authors.lastname: Rief
mesh_terms: D052898:Optical Tweezers; D058849:Protein Refolding; D058767:Protein Unfolding; D000072760:Single Molecule Imaging; D043222:Ubiquitin Thiolesterase / Q000737:chemistry*
chemical_list: D043222:Ubiquitin Thiolesterase
keywords: knotted proteins; optical tweezers; protein folding; single molecule; ubiquitin C-terminal hydrolase