Untangling the folding mechanism of the 5₂‐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 52‐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.

Structure and folding of a designed knotted protein

“…It was found that the threaded dimer folded about an order of magnitude more slowly than the wild-type protein, in which threading is not required during folding since both chains of the dimer are linear (22). Our observation of a complex energy landscape for 2ouf-knot is also consistent with recent experimental investigations of the folding pathways of naturally knotted proteins (11,23); all three of the proteins studied have been found to fold very slowly, with complex kinetic behaviors involving multiple intermediates. The lack of unknotted controls for those proteins has prevented direct assessment of the role played by their knotted topologies.…”

The Effect of Parkinson's-Disease-Associated Mutations on the Deubiquitinating Enzyme UCH-L1

“…The unfolding kinetics of wild-type and mutant UCH-L1 were measured by stopped-flow techniques for shorter timescales or by manual mixing with a fluorometer for longer timescales, as previously described 20 (further details are available in Supporting Information).…”

Characterization of the Folding of a 5 2 -Knotted Protein Using Engineered Single-Tryptophan Variants

“…Earlier experimental investigations of the folding of UCH-L3, a homolog of UCH-L1, showed that it folds via parallel pathways, each of which exhibits a hyperfluorescent intermediate state (27). Researchers have investigated wildtype (WT) UCH-L1 using a range of different techniques.…”