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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

Limited proteolysis of the isolated JD and ataxin-3 (14Q) following incubation of 10 μM protein with 0.1 μM trypsin in 10 mM ammonium acetate, pH 7.4, at 37 °C for 15 min. The major products of limited proteolysis of the JD domain (0–182, 60–182, 48–182, 0–47 + 60–182) are highlighted in (a) on JD structures in the form of ribbon diagrams, taken from the NMR structure for the isolated JD (PDB 1YZB [29]); these products resulted from cleavage at Arg47 and Arg59, both within the α2/α3 hairpin. The amino acid sequence for ataxin-3 (14Q) is shown in (b) with the major products of limited proteolysis (0–182, 60–182, 48–182, 0–47 + 60–182, 0–190 and 48–190). The JD domain (0–182) is highlighted in grey. The trypsin cleavage sites (orange bars) highlighted within the tail region (residues 183–361) all showed evidence of proteolysis. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
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fig0030: Limited proteolysis of the isolated JD and ataxin-3 (14Q) following incubation of 10 μM protein with 0.1 μM trypsin in 10 mM ammonium acetate, pH 7.4, at 37 °C for 15 min. The major products of limited proteolysis of the JD domain (0–182, 60–182, 48–182, 0–47 + 60–182) are highlighted in (a) on JD structures in the form of ribbon diagrams, taken from the NMR structure for the isolated JD (PDB 1YZB [29]); these products resulted from cleavage at Arg47 and Arg59, both within the α2/α3 hairpin. The amino acid sequence for ataxin-3 (14Q) is shown in (b) with the major products of limited proteolysis (0–182, 60–182, 48–182, 0–47 + 60–182, 0–190 and 48–190). The JD domain (0–182) is highlighted in grey. The trypsin cleavage sites (orange bars) highlighted within the tail region (residues 183–361) all showed evidence of proteolysis. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

Mentions: Both the JD and ataxin-3 (14Q) were subjected to limited proteolysis using the enzyme bovine trypsin to determine which regions of these proteins are accessible to this protease. Limited proteolysis is a powerful tool that can be used to gain additional structural information regarding a protein's solvent-exposed regions or its domain organisation. ESI-MS spectra obtained following the limited proteolysis of the JD and ataxin-3 (14Q) showed that many of the major products of limited proteolysis are the same for the corresponding regions of both species (Fig. 4). The major products of limited proteolysis observed for the JD are highlighted in Fig. 5a, and a comparison of the products obtained for the JD and the corresponding region of ataxin-3 (14Q) is presented in Table 1.


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)

Limited proteolysis of the isolated JD and ataxin-3 (14Q) following incubation of 10 μM protein with 0.1 μM trypsin in 10 mM ammonium acetate, pH 7.4, at 37 °C for 15 min. The major products of limited proteolysis of the JD domain (0–182, 60–182, 48–182, 0–47 + 60–182) are highlighted in (a) on JD structures in the form of ribbon diagrams, taken from the NMR structure for the isolated JD (PDB 1YZB [29]); these products resulted from cleavage at Arg47 and Arg59, both within the α2/α3 hairpin. The amino acid sequence for ataxin-3 (14Q) is shown in (b) with the major products of limited proteolysis (0–182, 60–182, 48–182, 0–47 + 60–182, 0–190 and 48–190). The JD domain (0–182) is highlighted in grey. The trypsin cleavage sites (orange bars) highlighted within the tail region (residues 183–361) all showed evidence of proteolysis. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
© Copyright Policy
Related In: Results  -  Collection

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

fig0030: Limited proteolysis of the isolated JD and ataxin-3 (14Q) following incubation of 10 μM protein with 0.1 μM trypsin in 10 mM ammonium acetate, pH 7.4, at 37 °C for 15 min. The major products of limited proteolysis of the JD domain (0–182, 60–182, 48–182, 0–47 + 60–182) are highlighted in (a) on JD structures in the form of ribbon diagrams, taken from the NMR structure for the isolated JD (PDB 1YZB [29]); these products resulted from cleavage at Arg47 and Arg59, both within the α2/α3 hairpin. The amino acid sequence for ataxin-3 (14Q) is shown in (b) with the major products of limited proteolysis (0–182, 60–182, 48–182, 0–47 + 60–182, 0–190 and 48–190). The JD domain (0–182) is highlighted in grey. The trypsin cleavage sites (orange bars) highlighted within the tail region (residues 183–361) all showed evidence of proteolysis. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
Mentions: Both the JD and ataxin-3 (14Q) were subjected to limited proteolysis using the enzyme bovine trypsin to determine which regions of these proteins are accessible to this protease. Limited proteolysis is a powerful tool that can be used to gain additional structural information regarding a protein's solvent-exposed regions or its domain organisation. ESI-MS spectra obtained following the limited proteolysis of the JD and ataxin-3 (14Q) showed that many of the major products of limited proteolysis are the same for the corresponding regions of both species (Fig. 4). The major products of limited proteolysis observed for the JD are highlighted in Fig. 5a, and a comparison of the products obtained for the JD and the corresponding region of ataxin-3 (14Q) is presented in Table 1.

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