Projects per year
Abstract
Based on extensive structural analysis it was proposed that RING E3 ligases prime the E2~ubiquitin conjugate (E2~Ub) for catalysis by locking it into a closed conformation where ubiquitin is folded back onto the E2 exposing the restrained thioester bond to attack by substrate nucleophile. However the proposal that the RING dependent closed conformation of E2~Ub represents the active form that mediates ubiquitin transfer has yet to be experimentally tested. To test this hypothesis we use single molecule Förster Resonance Energy Transfer (smFRET) to measure the conformation of a FRET labelled E2~Ub conjugate, which distinguishes between closed andalternative conformations. We describe a real-time FRET assay with a thioester linked E2~Ub conjugate to monitor single ubiquitination events and demonstrate that ubiquitin is transferred to substrate from the closed conformation. These findings are likely to be relevant to all RING E3 catalysed reactions ligating ubiquitin and other ubiquitin-like proteins (Ubls) to substrates.
Original language | English |
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Article number | 2846 |
Number of pages | 11 |
Journal | Nature Communications |
Volume | 11 |
DOIs | |
Publication status | Published - 5 Jun 2020 |
Keywords
- Enzyme mechanisms
- Single-molecule biophysics
- Ubiquitylation
ASJC Scopus subject areas
- General Physics and Astronomy
- General Chemistry
- General Biochemistry,Genetics and Molecular Biology
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Establishing the Basis for Selectivity in the SUMO Spray (Investigator Award)
Hay, R. (Investigator)
1/02/20 → 31/01/27
Project: Research
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Harnessing the PML-SUMO-RNF4 Axis for Cancer Therapy (Programme Grant)
Hay, R. (Investigator)
1/12/16 → 30/09/22
Project: Research
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Determining the Role and Mechanism of Action of SUMO Targeted Ubiquitin Ligase RNF4 in Maintaining Genome Integrity (Senior Investigator Award)
Hay, R. (Investigator)
1/10/12 → 31/01/20
Project: Research