title: The Knotted Protein UCH-L1 Exhibits Partially Unfolded Forms under Native Conditions that Share Common Structural Features with Its Kinetic Folding Intermediates.
abstract: The human ubiquitin C-terminal hydrolase, UCH-L1, is an abundant neuronal deubiquitinase that is associated with Parkinson's disease. It contains a complex Gordian knot topology formed by the polypeptide chain alone. Using a combination of fluorescence-based kinetic measurements, we show that UCH-L1 has two distinct kinetic folding intermediates that are transiently populated on parallel pathways between the denatured and native states. NMR hydrogen-deuterium exchange (HDX) experiments indicate the presence of partially unfolded forms (PUFs) of UCH-L1 under native conditions. HDX measurements as a function of urea concentration were used to establish the structure of the PUFs and pulse-labelled HDX NMR was used to show that the PUFs and the folding intermediates are likely the same species. In both cases, a similar stable core encompassing most of the central β-sheet is highly structured and α-helix 3, which is partially formed, packs against it. In contrast to the stable β-sheet core, the peripheral α-helices display significant local fluctuations leading to rapid exchange. The results also suggest that the main difference between the two kinetic intermediates is structure and packing of α-helices 3 and 7 and the degree of structure in β-strand 5. Together, the fluorescence and NMR results establish that UCH-L1 neither folds through a continuum of pathways nor by a single discrete pathway. Its folding is complex, the β-sheet core forms early and is present in both intermediate states, and the rate-limiting step which is likely to involve the threading of the chain to form the 52-knot occurs late on the folding pathway.
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK; Institute of Biological Chemistry, Academia Sinica, 128, Section 2, Academia Road, Taipei 11529, Taiwan.
authors.forename: Shih-Chi
authors.initials: SC
authors.lastname: Lou
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
authors.forename: Svava
authors.initials: S
authors.lastname: Wetzel
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
authors.forename: Hongyu
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
authors.forename: Elizabeth W
authors.initials: EW
authors.lastname: Crone
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, 128, Section 2, Academia Road, Taipei 11529, Taiwan; Institute of Biochemical Sciences, National Taiwan University, 1, Section 4, Roosevelt Road, Taipei 106, Taiwan.
authors.forename: Yun-Tzai
authors.initials: YT
authors.lastname: Lee
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK. Electronic address: sej13@cam.ac.uk.
authors.forename: Sophie E
authors.initials: SE
authors.lastname: Jackson
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK; Institute of Biological Chemistry, Academia Sinica, 128, Section 2, Academia Road, Taipei 11529, Taiwan; Institute of Biochemical Sciences, National Taiwan University, 1, Section 4, Roosevelt Road, Taipei 106, Taiwan. Electronic address: sthsu@gate.sinica.edu.tw.
authors.forename: Shang-Te Danny
authors.initials: SD
authors.lastname: Hsu
mesh_terms: D003903:Deuterium / Q000378:metabolism; D041961:Deuterium Exchange Measurement / Q000379:methods; D006801:Humans; D006859:Hydrogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D000072756:Protein Conformation, alpha-Helical / Q000502:physiology; D000072757:Protein Conformation, beta-Strand / Q000502:physiology; D011489:Protein Denaturation; D017510:Protein Folding; D043222:Ubiquitin Thiolesterase / Q000378:metabolism*
chemical_list: C482860:UCHL1 protein, human; D006859:Hydrogen; D003903:Deuterium; D043222:Ubiquitin Thiolesterase
keywords: folding intermediate; folding pathway; knotted protein; partially unfolded form; ubiquitin C-terminal hydrolase