MassIVE MSV000079990

Imported Reanalysis Dataset Public PXD004059

Mass spectrometry insights into a tandem ubiquitinâ??binding domain hybrid engineered for the selective recognition of unanchored polyubiquitin

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Description

Unanchored polyubiquitin chains are emerging as important regulators of cellular physiology with diverse roles paralleling those of substrate-conjugated polyubiquitin. However tools able to discriminate unanchored polyubiquitin chains of different isopeptide linkages have not been described. We describe the design of a linker-optimised ubiquitin-binding domain hybrid (t-UBD) containing two UBDs, a ZnF-UBP domain in tandem with a linkage-selective UBA domain, which exploits avidity effects to afford selective recognition of unanchored Lys48-linked polyubiquitin chains. Utilising native MS to quantitatively probe binding affinities we confirm cooperative binding of the UBDs within the synthetic protein, and desired binding specificity for Lys48-linked ubiquitin dimers. Furthermore MS/MS analyses indicate that the t-UBD, when applied as an affinity enrichment reagent, can be used to favour the purification of endogenous unanchored Lys48-linked polyubiquitin chains from mammalian cell extracts. Our study indicates that strategies for the rational design and engineering of polyubiquitin chain-selective binding in non-biological polymers are possible, paving the way for the generation of reagents to probe unanchored polyubiquitin chains of different linkages and more broadly the â??ubiquitomeâ??. [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Ubiquitin ; polyubiquitin

Contact

Principal Investigators:
(in alphabetical order) 		Dr Rob Layfield
Submitting User: ccms

Publications

Scott D, Garner TP, Long J, Strachan J, Mistry SC, Bottrill AR, Tooth DJ, Searle MS, Oldham NJ, Layfield R.
Mass spectrometry insights into a tandem ubiquitin-binding domain hybrid engineered for the selective recognition of unanchored polyubiquitin.
Proteomics. 2016 Jul;16(14):1961-1969.

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