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A tale of a tail: Structural insights into the conformational properties of the polyglutamine protein ataxin-3.

Scarff CA, Sicorello A, Tomé RJ, Macedo-Ribeiro S, Ashcroft AE, Radford SE - Int J Mass Spectrom (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.

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 states in vitro conferred 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.

No MeSH data available.


Related in: MedlinePlus

ESI-MS spectra of (a) the isolated Josephin domain (JD) and (b) ataxin-3 (14Q). Insets show the expanded (8×) region m/z 1000–2500. M, monomer; D, dimer.
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fig0015: ESI-MS spectra of (a) the isolated Josephin domain (JD) and (b) ataxin-3 (14Q). Insets show the expanded (8×) region m/z 1000–2500. M, monomer; D, dimer.

Mentions: The extent of protein ionisation during the ESI process is correlated directly with the surface-exposed area and mass of the protein [40]. The lowest charge states detected within the m/z spectrum for a protein are most reflective of the native structure of that protein, whilst partially folded protein conformations exhibit intermediate charge states and denatured or unfolded proteins carry the highest number of charges. In the case of the JD, the expected average charge states, based on mass [40], under native conditions and denaturing conditions are 9+ and 22+, respectively. For ataxin-3 (14Q) these are 14+ and 36+. The ESI-MS spectra obtained for the isolated JD (measured mass 21,092.8 Da; calculated mass 21,093.0 Da) and ataxin-3 (14Q) (measured mass 41,309.0 Da; calculated mass 41,307.2 Da) are shown in Fig. 2. One narrow charge state distribution, corresponding to monomeric 6+ to 10+ charge state ions, was populated predominantly in the spectrum of the JD whereas a much broader range of charge states was observed in the spectrum of ataxin-3 (14Q). Both spectra were acquired under identical experimental conditions and comparison of the two spectra thus indicates that the JD adopts mainly a single, compact conformation whereas full-length ataxin-3 (14Q) adopts multiple conformations, some more compact (11+ to 15+) and others more extended (18+ to 38+). The observation of such a wide array of charge states for a protein analysed under native-like solvent conditions, as observed for ataxin-3 (14Q), is unusual but is typical for intrinsically disordered and semi-disordered proteins [41,42]. A second, very minor, charge state distribution (10+ to 17+) was also observed in the JD spectrum indicating the low population of a partially unfolded state (Fig. 2a, inset). It is interesting to note that a population of ions corresponding to a dimeric species was also observed in the spectrum of ataxin-3 (14Q). These were either not observed, or were only very weakly populated, in the spectrum of the isolated JD.


A tale of a tail: Structural insights into the conformational properties of the polyglutamine protein ataxin-3.

Scarff CA, Sicorello A, Tomé RJ, Macedo-Ribeiro S, Ashcroft AE, Radford SE - Int J Mass Spectrom (2013)

ESI-MS spectra of (a) the isolated Josephin domain (JD) and (b) ataxin-3 (14Q). Insets show the expanded (8×) region m/z 1000–2500. M, monomer; D, dimer.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4375668&req=5

fig0015: ESI-MS spectra of (a) the isolated Josephin domain (JD) and (b) ataxin-3 (14Q). Insets show the expanded (8×) region m/z 1000–2500. M, monomer; D, dimer.
Mentions: The extent of protein ionisation during the ESI process is correlated directly with the surface-exposed area and mass of the protein [40]. The lowest charge states detected within the m/z spectrum for a protein are most reflective of the native structure of that protein, whilst partially folded protein conformations exhibit intermediate charge states and denatured or unfolded proteins carry the highest number of charges. In the case of the JD, the expected average charge states, based on mass [40], under native conditions and denaturing conditions are 9+ and 22+, respectively. For ataxin-3 (14Q) these are 14+ and 36+. The ESI-MS spectra obtained for the isolated JD (measured mass 21,092.8 Da; calculated mass 21,093.0 Da) and ataxin-3 (14Q) (measured mass 41,309.0 Da; calculated mass 41,307.2 Da) are shown in Fig. 2. One narrow charge state distribution, corresponding to monomeric 6+ to 10+ charge state ions, was populated predominantly in the spectrum of the JD whereas a much broader range of charge states was observed in the spectrum of ataxin-3 (14Q). Both spectra were acquired under identical experimental conditions and comparison of the two spectra thus indicates that the JD adopts mainly a single, compact conformation whereas full-length ataxin-3 (14Q) adopts multiple conformations, some more compact (11+ to 15+) and others more extended (18+ to 38+). The observation of such a wide array of charge states for a protein analysed under native-like solvent conditions, as observed for ataxin-3 (14Q), is unusual but is typical for intrinsically disordered and semi-disordered proteins [41,42]. A second, very minor, charge state distribution (10+ to 17+) was also observed in the JD spectrum indicating the low population of a partially unfolded state (Fig. 2a, inset). It is interesting to note that a population of ions corresponding to a dimeric species was also observed in the spectrum of ataxin-3 (14Q). These were either not observed, or were only very weakly populated, in the spectrum of the isolated JD.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.

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 states in vitro conferred 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.

No MeSH data available.


Related in: MedlinePlus