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Methods for Structural Analysis of Amyloid Fibrils in Misfolding Diseases

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Protein Misfolding Diseases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1873))

Abstract

Many proteins and peptides are able to self-assemble in solution in vitro and in vivo to form amyloid-like fibrils. These fibrils share common structural characteristics. In order for a fibril to be characterized as amyloid, it is expected to fit certain criteria including the composition of cross-β. Here we describe how the formation of amyloid fibrils can be characterized in vitro using a variety of methods including circular dichroism and intrinsic tyrosine/tryptophan fluoresence to follow conformational changes; Thioflavin and/or ThS assembly to monitor nucleation and growth; transmission electron microscopy to visualize fibrillar morphology and X-ray fiber diffraction to examine cross-β structure.

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References

  1. Sipe JD, Benson MD, Buxbaum JN, Ikeda S, Merlini G, Saraiva MJ, Westermark P (2014) Nomenclature 2014: amyloid fibril proteins and clinical classification of the amyloidosis. Amyloid 21(4):221–224. https://doi.org/10.3109/13506129.2014.964858

    Article  PubMed  Google Scholar 

  2. Puchtler H, Sweat F (1965) Congo red as a stain for fluorescence microscopy of amyloid. J Histochem Cytochem 13(8):693–694

    Article  CAS  Google Scholar 

  3. LeVine H 3rd (1999) Quantification of beta-sheet amyloid fibril structures with thioflavin T. Methods Enzymol 309:274–284

    Article  CAS  Google Scholar 

  4. Glabe CG (2004) Conformation-dependent antibodies target diseases of protein misfolding. Trends Biochem Sci 29(10):542–547

    Article  CAS  Google Scholar 

  5. Lambert MP, Velasco PT, Chang L, Viola KL, Fernandez S, Lacor PN, Khuon D, Gong Y, Bigio EH, Shaw P, De Felice FG, Krafft GA, Klein WL (2007) Monoclonal antibodies that target pathological assemblies of Abeta. J Neurochem 100(1):23–35. https://doi.org/10.1111/j.1471-4159.2006.04157.x JNC4157 [pii]

    Article  CAS  PubMed  Google Scholar 

  6. Marshall KE, Hicks MR, Williams TL, Hoffmann SV, Rodger A, Dafforn TR, Serpell LC (2010) Characterizing the assembly of the Sup35 yeast prion fragment, GNNQQNY: structural changes accompany a fiber-to-crystal switch. Biophys J 98(2):330–338. https://doi.org/10.1016/j.bpj.2009.10.020 S0006-3495(09)01627-0 [pii]

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Sawaya MR, Sambashivan S, Nelson R, Ivanova MI, Sievers SA, Apostol MI, Thompson MJ, Balbirnie M, Wiltzius JJ, McFarlane HT, Madsen AO, Riekel C, Eisenberg D (2007) Atomic structures of amyloid cross-beta spines reveal varied steric zippers. Nature 447(7143):453–457

    Article  CAS  Google Scholar 

  8. Tycko R (2001) Biomolecular solid state NMR: advances in structural methodology and applications to peptide and protein fibrils. Annu Rev Phys Chem 52:575–606

    Article  CAS  Google Scholar 

  9. Fitzpatrick AWP, Falcon B, He S, Murzin AG, Murshudov G, Garringer HJ, Crowther RA, Ghetti B, Goedert M, Scheres SHW (2017) Cryo-EM structures of tau filaments from Alzheimer's disease. Nature 547(7662):185–190. https://doi.org/10.1038/nature23002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. van der Wel PC, Lewandowski JR, Griffin RG (2007) Solid-state NMR study of amyloid nanocrystals and fibrils formed by the peptide GNNQQNY from yeast prion protein Sup35p. J Am Chem Soc 129(16):5117–5130

    Article  Google Scholar 

  11. White HE, Hodgkinson JL, Jahn TR, Cohen-Krausz S, Gosal WS, Muller S, Orlova EV, Radford SE, Saibil HR (2009) Globular tetramers of beta(2)-microglobulin assemble into elaborate amyloid fibrils. J Mol Biol 389(1):48–57

    Article  CAS  Google Scholar 

  12. Xue WF, Radford SE (2013) An imaging and systems modeling approach to fibril breakage enables prediction of amyloid behavior. Biophys J 105(12):2811–2819. https://doi.org/10.1016/j.bpj.2013.10.034

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Goldsbury C, Kistler J, Aebi U, Arvinte T, Cooper GJ (1999) Watching amyloid fibrils grow by time-lapse atomic force microscopy. J Mol Biol 285(1):33–39

    Article  CAS  Google Scholar 

  14. CCP4 (1994) The CCP4 suite programs for crystallography. Acta Cryst D50:760–763

    Google Scholar 

  15. Hammersley AP (1998) FIT2D V9.129 Reference Manual V3.1. ESRF Internal Report ESRF98HA01T

    Google Scholar 

  16. Makin OS, Sikorski P, Serpell LC (2007) CLEARER: a new tool for the analysis of X-ray fibre diffraction patterns and diffraction simulation from atomic structural models. Appl Cryst 40(5):966–972

    Article  Google Scholar 

  17. Broersen K, Jonckheere W, Rozenski J, Vandersteen A, Pauwels K, Pastore A, Rousseau F, Schymkowitz J (2011) A standardized and biocompatible preparation of aggregate-free amyloid beta peptide for biophysical and biological studies of Alzheimer's disease. Protein Eng Des Sel 24(9):743–750. https://doi.org/10.1093/protein/gzr020

    Article  CAS  PubMed  Google Scholar 

  18. Bitan G, Frandinger EA, Spring S, Teplow D (2005) Neurotoxic protein oligomers- what you see is not always what you get. Amyloid 2(Jun):88–95

    Article  Google Scholar 

  19. Fezoui Y, Hartley DM, Harper JD, Khurana R, Walsh DM, Condron MM, Selkoe DJ, Lansbury PT Jr, Fink AL, Teplow DB (2000) An improved method of preparing the amyloid beta-protein for fibrillogenesis and neurotoxicity experiments. Amyloid 7(3):166–178

    Article  CAS  Google Scholar 

  20. Kelly SM, Jess TJ, Price NC (2005) How to study proteins by circular dichroism. Biochim Biophys Acta 1751(2):119–139. https://doi.org/10.1016/j.bbapap.2005.06.005

    Article  CAS  Google Scholar 

  21. Morris KL, Rodger A, Hicks MR, Debulpaep M, Schymkowitz J, Rousseau F, Serpell LC (2013) Exploring the sequence-structure relationship for amyloid peptides. Biochem J 450(2):275–283. https://doi.org/10.1042/BJ20121773

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Dafforn TR, Rajendra J, Halsall DJ, Serpell LC, Rodger A (2004) Protein fiber linear dichroism for structure determination and kinetics in a low-volume, low-wavelength couette flow cell. Biophys J 86(1 Pt 1):404–410

    Article  CAS  Google Scholar 

  23. Rodger A, Marrington R, Geeves MA, Hicks M, de Alwis L, Halsall DJ, Dafforn TR (2006) Looking at long molecules in solution: what happens when they are subjected to Couette flow? Phys Chem Chem Phys 8(27):3161–3171

    Article  CAS  Google Scholar 

  24. Rodger A, Norden B (1997) Circular Dichroism and linear Dichroism. Oxford University Press, Oxford

    Google Scholar 

  25. Morris KL, Serpell LC (2012) X-ray fibre diffraction studies of amyloid fibrils. Methods Mol Biol 849:121–135. https://doi.org/10.1007/978-1-61779-551-0_9

    Article  CAS  PubMed  Google Scholar 

  26. Winn MD (2003) An overview of the CCP4 project in protein crystallography: an example of a collaborative project. J Synchrotron Radiat 10. (Pt 1:23–25

    Article  CAS  Google Scholar 

  27. Makin OS, Serpell LC (2005) X-ray diffraction studies of amyloid structure. In: Sigurdsson EM (ed) Amyloid proteins: methods and protocols. Humana press, Totowa, NJ, pp 67–80

    Google Scholar 

  28. Al-Hilaly YK, Biasetti L, Blakeman BJ, Pollack SJ, Zibaee S, Abdul-Sada A, Thorpe JR, Xue WF, Serpell LC (2016) The involvement of dityrosine crosslinking in alpha-synuclein assembly and deposition in Lewy bodies in Parkinson's disease. Sci Rep 6:39171. https://doi.org/10.1038/srep39171

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Author information

Authors and Affiliations

  1. School of Life Sciences, University of Sussex, East Sussex, UK

    Devkee M. Vadukul, Youssra K. Al-Hilaly & Louise C. Serpell

  2. Department of Chemistry, College of Sciences, Al-Mustansiriyah University, Baghdad, Iraq

    Youssra K. Al-Hilaly

Authors
  1. Devkee M. Vadukul
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  2. Youssra K. Al-Hilaly
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  3. Louise C. Serpell
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Corresponding author

Correspondence to Louise C. Serpell .

Editor information

Editors and Affiliations

  1. BioISI – Biosystems and Integrative Sciences Institute, Faculty of Sciences University of Lisbon, Lisbon, Portugal

    Cláudio M. Gomes

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Vadukul, D.M., Al-Hilaly, Y.K., Serpell, L.C. (2019). Methods for Structural Analysis of Amyloid Fibrils in Misfolding Diseases. In: Gomes, C. (eds) Protein Misfolding Diseases. Methods in Molecular Biology, vol 1873. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8820-4_7

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  • DOI: https://doi.org/10.1007/978-1-4939-8820-4_7

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8819-8

  • Online ISBN: 978-1-4939-8820-4

  • eBook Packages: Springer Protocols

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