Production and characterisation of modularly deuterated UBE2D1-Ub conjugate by small angle neutron and X-ray scattering
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Date
2022-10-26
Journal Title
Journal ISSN
Volume Title
Publisher
Springer
Abstract
This structural study exploits the possibility to use modular protein deuteration to facilitate the study of ubiquitin signalling, transfer, and modification. A protein conjugation reaction is used to combine protonated E2 enzyme with deuterated ubiquitin for small angle X-ray and neutron scattering with neutron contrast variation. The combined biomolecules stay as a monodisperse system during data collection in both protonated and deuterated buffers indicating long stability of the E2–Ub conjugate. With multiphase ab initio shape restoration and rigid body modelling, we reconstructed the shape of a E2–Ub-conjugated complex of UBE2D1 linked to ubiquitin via an isopeptide bond. Solution X-ray and neutron scattering data for this E2–Ub conjugate in the absence of E3 jointly indicate an ensemble of open and backbent states, with a preference for the latter in solution. The approach of combining protonated and labelled proteins can be used for solution studies to assess localization and movement of ubiquitin and could be widely applied to modular Ub systems in general. © The Author(s) 2022. Open Access CC-BY
Description
We acknowledge the support and assistance of the staff at the Australian Centre for Neutron Scattering and the National Deuteration Facility, proposal codes NDF7226 and P7227. The National Deuteration Facility is partly supported by the National Collaborative Research Infrastructure Strategy–an initiative of the Australian Government.
Keywords
Proteins, Enzymes, Modifications, Scattering, Data, Solutions
Citation
Pietras, Z, Duff, A. P., Morad V., Wood, K., Jeffries, C. M., & Sunnerhagen, M. (2022). Production and characterisation of modularly deuterated UBE2D1-Ub conjugate by small angle neutron and x-ray scattering. European Biophysics Journal, 51(7-8), 569-577. doi: 10.1007/s00249-022-01620-1