Cross-β-Sheet Supersecondary Structure in Amyloid Folds: Techniques for Detection and Characterization

  • Raimon Sabaté
  • Salvador Ventura
Part of the Methods in Molecular Biology book series (MIMB, volume 932)


The formation of protein aggregates is linked to the onset of several human disorders of increasing prevalence, ranging from dementia to diabetes. In most of these diseases, the toxic effect is exerted by the self-assembly of initially soluble proteins into insoluble amyloid-like fibrils. Independently of the protein origin, all these macromolecular assemblies share a common supersecondary structure: the cross-β-sheet conformation, in which a core of β-strands is aligned perpendicularly to the fibril axis forming extended regular β-sheets. Due to this ubiquity, the presence of cross-β-sheet conformational signatures is usually exploited to detect, characterize, and screen for amyloid fibrils in protein samples. Here we describe in detail some of the most commonly used methods to analyze such supersecondary structure.

Key words

Amyloid Beta-fold Fibril Cross-beta-sheet Protein aggregation 



This work was supported by grants BFU2010-14901 from Ministerio de Ciencia e Innovación (Spain), 2009-SGR-760 and 2009-CTP-00004 from AGAUR (Generalitat de Catalunya). SV has been granted an ICREA Academia award (ICREA).


  1. 1.
    de Groot NS, Sabate R, Ventura S (2009) Amyloids in bacterial inclusion bodies. Trends Biochem Sci 34(8):408–416PubMedCrossRefGoogle Scholar
  2. 2.
    Jahn TR, Radford SE (2008) Folding versus aggregation: polypeptide conformations on competing pathways. Arch Biochem Biophys 469(1):100–117PubMedCrossRefGoogle Scholar
  3. 3.
    Dasari M, Espargaro A, Sabate R et al (2011) Bacterial inclusion bodies of Alzheimer’s disease beta-amyloid peptides can be employed to study native-like aggregation intermediate states. Chembiochem 12(3):407–423PubMedCrossRefGoogle Scholar
  4. 4.
    Hubbell WL, Cafiso DS, Altenbach C (2000) Identifying conformational changes with site-directed spin labeling. Nat Struct Biol 7(9):735–739. doi: 10.1038/78956 PubMedCrossRefGoogle Scholar
  5. 5.
    Pelczer I, Carter BG (1997) Data processing in multidimensional NMR. Methods Mol Biol 60:71–155PubMedGoogle Scholar
  6. 6.
    Sawaya MR, Sambashivan S, Nelson R et al (2007) Atomic structures of amyloid cross-beta spines reveal varied steric zippers. Nature 447(7143):453–457PubMedCrossRefGoogle Scholar
  7. 7.
    Tycko R (2006) Molecular structure of amyloid fibrils: insights from solid-state NMR. Q Rev Biophys 39(1):1–55PubMedCrossRefGoogle Scholar
  8. 8.
    Tycko R (2011) Solid-state NMR studies of amyloid fibril structure. Annu Rev Phys Chem 62:279–299PubMedCrossRefGoogle Scholar
  9. 9.
    Wasmer C, Lange A, Van Melckebeke H et al (2008) Amyloid fibrils of the HET-s(218–289) prion form a beta solenoid with a triangular hydrophobic core. Science 319(5869):1523–1526PubMedCrossRefGoogle Scholar
  10. 10.
    Groenning M, Olsen L, van de Weert M et al (2007) Study on the binding of Thioflavin T to beta-sheet-rich and non-beta-sheet cavities. J Struct Biol 158(3):358–369PubMedCrossRefGoogle Scholar
  11. 11.
    Zhavoronkov N, Gritsai Y, Bargheer M et al (2005) Microfocus Cu K(alpha) source for femtosecond X-ray science. Opt Lett 30(13):1737–1739PubMedCrossRefGoogle Scholar
  12. 12.
    Makin O, Sikorski P, Serpell L (2007) CLEARER: a new tool for the analysis of X-ray fibre diffraction patterns and diffraction simulation from atomic structural models. J Appl Crystallogr 40:966–972CrossRefGoogle Scholar
  13. 13.
    Steensma DP (2001) “Congo” red: out of Africa? Arch Pathol Lab Med 125(2):250–252PubMedGoogle Scholar
  14. 14.
    Klunk WE, Pettegrew JW, Abraham DJ (1989) Two simple methods for quantifying low-affinity dye-substrate binding. J Histochem Cytochem 37(8):1293–1297PubMedCrossRefGoogle Scholar
  15. 15.
    Klunk WE, Pettegrew JW, Abraham DJ (1989) Quantitative evaluation of Congo red binding to amyloid-like proteins with a beta-pleated sheet conformation. J Histochem Cytochem 37(8):1273–1281PubMedCrossRefGoogle Scholar
  16. 16.
    Kodali R, Wetzel R (2007) Polymorphism in the intermediates and products of amyloid assembly. Curr Opin Struct Biol 17(1):48–57PubMedCrossRefGoogle Scholar
  17. 17.
    Klunk WE, Jacob RF, Mason RP (1999) Quantifying amyloid by Congo red spectral shift assay. Methods Enzymol 309:285–305PubMedCrossRefGoogle Scholar
  18. 18.
    Klunk WE, Jacob RF, Mason RP (1999) Quantifying amyloid beta-peptide (Abeta) aggregation using the Congo red-Abeta (CR-abeta) spectrophotometric assay. Anal Biochem 266(1):66–76PubMedCrossRefGoogle Scholar
  19. 19.
    Inouye H, Nguyen JT, Fraser PE et al (2000) Histidine residues underlie Congo red binding to A beta analogs. Amyloid 7(3):179–188PubMedCrossRefGoogle Scholar
  20. 20.
    Sabate R, Estelrich J (2003) Pinacyanol as effective probe of fibrillar beta-amyloid peptide: comparative study with Congo red. Biopolymers 72(6):455–463PubMedCrossRefGoogle Scholar
  21. 21.
    Schutz AK, Soragni A, Hornemann S et al (2011) The amyloid-Congo red interface at atomic resolution. Angew Chem Int Ed Engl. doi: 10.1002/anie.201008276
  22. 22.
    Sabate R, Espargaro A, Saupe SJ et al (2009) Characterization of the amyloid bacterial inclusion bodies of the HET-s fungal prion. Microb Cell Fact 8:56PubMedCrossRefGoogle Scholar
  23. 23.
    Puchtler H, Sweat F (1965) Congo red as a stain for fluorescence microscopy of amyloid. J Histochem Cytochem 13(8):693–694PubMedCrossRefGoogle Scholar
  24. 24.
    Giorgadze TA, Shiina N, Baloch ZW et al (2004) Improved detection of amyloid in fat pad aspiration: an evaluation of Congo red stain by fluorescent microscopy. Diagn Cytopathol 31(5):300–306PubMedCrossRefGoogle Scholar
  25. 25.
    LeVine H 3rd (1999) Quantification of beta-sheet amyloid fibril structures with thioflavin T. Methods Enzymol 309:274–284PubMedCrossRefGoogle Scholar
  26. 26.
    Naiki H, Gejyo F (1999) Kinetic analysis of amyloid fibril formation. Methods Enzymol 309:305–318PubMedCrossRefGoogle Scholar
  27. 27.
    Sabate R, Lascu I, Saupe SJ (2008) On the binding of Thioflavin-T to HET-s amyloid fibrils assembled at pH 2. J Struct Biol 162(3):387–396PubMedCrossRefGoogle Scholar
  28. 28.
    Dzwolak W, Pecul M (2005) Chiral bias of amyloid fibrils revealed by the twisted conformation of Thioflavin T: an induced circular dichroism/DFT study. FEBS Lett 579(29):6601–6603PubMedCrossRefGoogle Scholar
  29. 29.
    Sabate R, Saupe SJ (2007) Thioflavin T fluorescence anisotropy: an alternative technique for the study of amyloid aggregation. Biochem Biophys Res Commun 360(1):135–138PubMedCrossRefGoogle Scholar
  30. 30.
    Chiti F, Dobson CM (2006) Protein misfolding, functional amyloid, and human disease. Annu Rev Biochem 75:333–366PubMedCrossRefGoogle Scholar
  31. 31.
    Bouchard M, Zurdo J, Nettleton EJ et al (2000) Formation of insulin amyloid fibrils followed by FTIR simultaneously with CD and electron microscopy. Protein Sci 9(10):1960–1967PubMedCrossRefGoogle Scholar
  32. 32.
    de Groot NS, Parella T, Aviles FX et al (2007) Ile-phe dipeptide self-assembly: clues to amyloid formation. Biophys J 92(5):1732–1741PubMedCrossRefGoogle Scholar
  33. 33.
    Sabate R, Espargaro A, de Groot NS et al (2010) The role of protein sequence and amino acid composition in amyloid formation: scrambling and backward reading of IAPP amyloid fibrils. J Mol Biol 404(2):337–352PubMedCrossRefGoogle Scholar
  34. 34.
    Madine J, Jack E, Stockley PG et al (2008) Structural insights into the polymorphism of amyloid-like fibrils formed by region 20–29 of amylin revealed by solid-state NMR and X-ray fiber diffraction. J Am Chem Soc 130(45):14990–15001PubMedCrossRefGoogle Scholar
  35. 35.
    Morris K, Serpell L (2010) From natural to designer self-assembling biopolymers, the structural characterisation of fibrous proteins & peptides using fibre diffraction. Chem Soc Rev 39(9):3445–3453PubMedCrossRefGoogle Scholar
  36. 36.
    Hubbard SJ (1998) The structural aspects of limited proteolysis of native proteins. Biochim Biophys Acta 1382(2):191–206PubMedCrossRefGoogle Scholar
  37. 37.
    Collins SR, Douglass A, Vale RD et al (2004) Mechanism of prion propagation: amyloid growth occurs by monomer addition. PLoS Biol 2(10):e321PubMedCrossRefGoogle Scholar
  38. 38.
    Jarrett JT, Lansbury PT Jr (1993) Seeding “one-dimensional crystallization” of amyloid: a pathogenic mechanism in Alzheimer’s disease and scrapie? Cell 73(6):1055–1058PubMedCrossRefGoogle Scholar
  39. 39.
    Sabate R, Gallardo M, Estelrich J (2003) An autocatalytic reaction as a model for the kinetics of the aggregation of beta-amyloid. Biopolymers 71(2):190–195PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  1. 1.Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia MolecularUniversitat Autònoma de BarcelonaBellaterraSpain

Personalised recommendations