Production and characterisation of modularly deuterated UBE2D1-Ub conjugate by small angle neutron and X-ray scattering

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dc.contributor.authorPietras, Zen_AU
dc.contributor.authorDuff, APen_AU
dc.contributor.authorMorad, Ven_AU
dc.contributor.authorWood, Ken_AU
dc.contributor.authorJeffries, CMen_AU
dc.contributor.authorSunnerhagen, Men_AU
dc.date.accessioned2023-04-28T05:45:23Zen_AU
dc.date.available2023-04-28T05:45:23Zen_AU
dc.date.issued2022-10-26en_AU
dc.date.statistics2023-04-19en_AU
dc.descriptionWe 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.en_AU
dc.description.abstractThis 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-BYen_AU
dc.description.sponsorshipThis work was supported by the Swedish Foundation for Strategic Research by a PhD position (ZP) within the national graduate school SwedNess (GSn15-0008), and by a Swedish Research Council (VR) project grant (2018-04392) in support of neutron research (MS). Open access funding provided by Linköping University.en_AU
dc.identifier.citationPietras, 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/s00249-022-01620-1en_AU
dc.identifier.issn0175-7571en_AU
dc.identifier.issue7-8en_AU
dc.identifier.journaltitleEuropean Biophysics Journalen_AU
dc.identifier.pagination569-577en_AU
dc.identifier.urihttps://link.springer.com/article/10.1007/s00249-022-01620-1en_AU
dc.identifier.urihttps://apo.ansto.gov.au/dspace/handle/10238/14943en_AU
dc.identifier.volume51en_AU
dc.language.isoenen_AU
dc.publisherSpringeren_AU
dc.subjectProteinsen_AU
dc.subjectEnzymesen_AU
dc.subjectModificationsen_AU
dc.subjectScatteringen_AU
dc.subjectDataen_AU
dc.subjectSolutionsen_AU
dc.titleProduction and characterisation of modularly deuterated UBE2D1-Ub conjugate by small angle neutron and X-ray scatteringen_AU
dc.typeJournal Articleen_AU
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