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Updated: Aug 24, 2026

In Vitro Analysis of E3 Ubiquitin Ligase Function
Published on: May 14, 2021
Is an intermediate state populated on the folding pathway of ubiquitin?
Heather M Went1, Claudia G Benitez-Cardoza, Sophie E Jackson
1Chemistry Department, Centre for Protein Engineering, Lensfield Road, Cambridge CB2 1EW, UK.
Abstract:
In the last couple of years, there has been increasing debate as to the presence and role of intermediate states on the folding pathways of several small proteins, including the 76-residue protein ubiquitin. Here, we present detailed kinetic studies to establish whether an intermediate state is ever populated during the folding of this protein. We show that the differences observed in previous studies are attributable to the transient aggregation of the protein during folding. Using a highly soluble construct of ubiquitin, which does not aggregate during folding, we establish the conditions in which an intermediate state is sufficiently stable to be observed by kinetic measurements.
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