title: A tale of a tail: Structural insights into the conformational properties of the polyglutamine protein ataxin-3.
abstract: Ataxin-3 is the protein responsible for the neurodegenerative polyglutamine disease Spinocerebellar ataxia type 3. Full structural characterisation of ataxin-3 is required to aid in understanding the mechanism of disease. Despite extensive study, little is known about the conformational properties of the full-length protein, in either its non-expanded healthy or expanded pathogenic forms, particularly since its polyglutamine-containing region has denied structural elucidation. In this work, travelling-wave ion mobility spectrometry-mass spectrometry and limited proteolysis have been used to compare the conformational properties of full-length non-expanded ataxin-3 (14Q) and its isolated N-terminal Josephin domain (JD). Limited proteolysis experiments have confirmed that the JD is stable, being extremely resistant to trypsin digestion, with the exception of the α2/α3 hairpin which is flexible and exposed to protease cleavage in solution. The C-terminal region of ataxin-3 which contains the glutamine-rich sequences is largely unstructured, showing little resistance to limited proteolysis. Using ion mobility spectrometry-mass spectrometry we show that ataxin-3 (14Q) adopts a wide range of conformational statesin vitroconferred by the flexibility of its C-terminal tail and the α2/α3 hairpin of the N-terminal JD. This study highlights how the power of MS-based approaches to protein structural characterisation can be particularly useful when the target protein is aggregation-prone and has intrinsically unordered regions.
authors.affiliation: Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Charlotte A
authors.initials: CA
authors.lastname: Scarff
authors.affiliation: Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Sicorello
authors.affiliation: Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK ; IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal.
authors.forename: Ricardo J L
authors.initials: RJ
authors.lastname: Tomé
authors.affiliation: IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal.
authors.forename: Sandra
authors.initials: S
authors.lastname: Macedo-Ribeiro
authors.affiliation: Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Alison E
authors.initials: AE
authors.lastname: Ashcroft
authors.affiliation: Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Sheena E
authors.initials: SE
authors.lastname: Radford
keywords: Amyloid; Ataxin-3; Electrospray ionisation-mass spectrometry; Ion mobility spectrometry; PolyQ
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