Lou, S-C;
Wetzel, S;
Zhang, H;
Crone, EW;
Lee, Y-T;
Jackson, SE;
Hsu, S-TD;
(2016)
The Knotted Protein UCH-L1 Exhibits Partially Unfolded Forms under Native Conditions that Share Common Structural Features with Its Kinetic Folding Intermediates.
Journal of Molecular Biology
, 428
(11)
pp. 2507-2520.
10.1016/j.jmb.2016.04.002.
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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.
| Type: | Article |
|---|---|
| Title: | The Knotted Protein UCH-L1 Exhibits Partially Unfolded Forms under Native Conditions that Share Common Structural Features with Its Kinetic Folding Intermediates. |
| Location: | England |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1016/j.jmb.2016.04.002 |
| Language: | English |
| Additional information: | This version is the author accepted manuscript. For information on re-use, please refer to the publisher’s terms and conditions. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > UCL BEAMS UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10064539 |
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