title: Untangling the folding mechanism of the 5(2)-knotted protein UCH-L3.
abstract: Proteins possessing deeply embedded topological knots in their structure add a stimulating new challenge to the already complex protein-folding problem. The most complicated knotted topology observed to date belongs to the human enzyme ubiquitin C-terminal hydrolase UCH-L3, which is an integral part of the ubiquitin-proteasome system. The structure of UCH-L3 contains five distinct crossings of its polypeptide chain, and it adopts a 5(2)-knotted topology, making it a fascinating target for folding studies. Here, we provide the first in depth characterization of the stability and folding of UCH-L3. We show that the protein can unfold and refold reversibly in vitro without the assistance of molecular chaperones, demonstrating that all the information necessary for the protein to find its knotted native structure is encoded in the amino acid sequence, just as with any other globular protein, and that the protein does not enter into any deep kinetic traps. Under equilibrium conditions, the unfolding of UCH-L3 appears to be two-state, however, multiphasic folding and unfolding kinetics are observed and the data are consistent with a folding pathway in which two hyperfluorescent intermediates are formed. In addition, a very slow phase in the folding kinetics is shown to be limited by proline-isomerization events. Overall, the data suggest that a knotted topology, even in its most complex form, does not necessarily limit folding in vitro, however, it does seem to require a complex folding mechanism which includes the formation of several distinct intermediate species.
authors.affiliation: University Chemical Laboratory, Cambridge, UK.
authors.forename: Fredrik I
authors.initials: FI
authors.lastname: Andersson
authors.forename: David G
authors.initials: DG
authors.lastname: Pina
authors.forename: Anna L
authors.initials: AL
authors.lastname: Mallam
authors.forename: Georg
authors.initials: G
authors.lastname: Blaser
authors.forename: Sophie E
authors.initials: SE
authors.lastname: Jackson
mesh_terms: D002942:Circular Dichroism; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D011487:Protein Conformation; D017510:Protein Folding; D013816:Thermodynamics; D043222:Ubiquitin Thiolesterase
chemical_list: C509806:UCHL3 protein, human; D043222:Ubiquitin Thiolesterase; D003546:Cysteine Endopeptidases