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Polyglutamine aggregate structure in vitro and in vivo; new avenues for coherent anti-Stokes Raman scattering microscopy

Perney, Nicolas M., Braddick, Lucy, Jurna, Martin, Garbacik, Erik T., Offerhaus, Herman L., Serpell, Louise C., Blanch, Ewan, Holden-Dye, Lindy, Brocklesby, William S. and Melvin, Tracy (2012) Polyglutamine aggregate structure in vitro and in vivo; new avenues for coherent anti-Stokes Raman scattering microscopy PLoS ONE, 7, (7), e40536. (doi:10.1371/journal.pone.0040536).

Record type: Article


Coherent anti-Stokes Raman scattering (CARS) microscopy is applied for the first time for the evaluation of the protein secondary structure of polyglutamine (polyQ) aggregates in vivo. Our approach demonstrates the potential for translating information about protein structure that has been obtained in vitro by X-ray diffraction into a microscopy technique that allows the same protein structure to be detected in vivo. For these studies, fibres of polyQ containing peptides (D2Q15K2) were assembled in vitro and examined by electron microscopy and X-ray diffraction methods; the fibril structure was shown to be cross beta-sheet. The same polyQ fibres were evaluated by Raman spectroscopy and this further confirmed the beta-sheet structure, but indicated that the structure is highly rigid, as indicated by the strong Amide I signal at 1659 cm-1. CARS spectra were simulated using the Raman spectrum taking into account potential non-resonant contributions, providing evidence that the Amide I signal remains strong, but slightly shifted to lower wavenumbers. Combined CARS (1657 cm=1) and multi-photon fluorescence microscopy of chimeric fusions of yellow fluorescent protein (YFP) with polyQ (Q40) expressed in the body wall muscle cells of Caenorhabditis elegans nematodes (1 day old adult hermaphrodites) revealed diffuse and foci patterns of Q40-YFP that were both fluorescent and exhibited stronger CARS (1657 cm-1) signals than in surrounding tissues at the resonance for the cross beta-sheet polyQ in vitro.

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Published date: 20 July 2012
Organisations: Optoelectronics Research Centre, Electronics & Computer Science


Local EPrints ID: 341467
ISSN: 1932-6203
PURE UUID: 6e90b094-e594-4cf3-b1a1-712b0fd0a5ac
ORCID for William S. Brocklesby: ORCID iD

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Date deposited: 27 Jul 2012 08:44
Last modified: 18 Jul 2017 05:35

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Author: Nicolas M. Perney
Author: Lucy Braddick
Author: Martin Jurna
Author: Erik T. Garbacik
Author: Herman L. Offerhaus
Author: Louise C. Serpell
Author: Ewan Blanch
Author: Tracy Melvin

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