Advertisement

An Aggregate Weight-Normalized Thioflavin-T Measurement Scale for Characterizing Polymorphic Amyloids and Assembly Intermediates

  • Ronald Wetzel
  • Saketh Chemuru
  • Pinaki Misra
  • Ravi Kodali
  • Smita Mukherjee
  • Karunakar Kar
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1777)

Abstract

The red shift in the fluorescence excitation spectra of thioflavin dyes upon binding to fibrils has been a boon to the amyloid field, offering simple and effective methods for the qualitative detection of amyloid in tissue samples and for quantitation of particular fibril preparations with gravimetric linearity. The quantitative aspect of the thioflavin T (ThT) response, however, comes with an important caveat that bestows both significant limitations and great untapped power. It is now well established that amyloid fibrils of different proteins, as well as polymorphic fibrils of the same protein, can exhibit vastly different ThT fluorescence intensities for the same weight concentration of aggregates. Furthermore, the aggregated intermediates commonly observed in amyloid assembly reactions can exhibit aggregate weight-normalized (AWN) ThT fluorescence intensities that vary from essentially zero through a wide range of intermediate values before reaching the intensity of homogeneous, mature amyloid. These features make it very difficult to quantitatively interpret, without additional data, the time-dependent development of ThT fluorescence intensity in an assembly reaction. In this chapter, we describe a method for coupling ex situ ThT fluorescence determinations with an analytical HPLC supported sedimentation assay (also described in detail) that can provide significant new insights into amyloid assembly reactions. The time dependent aggregation data provided by the sedimentation assay reveals a time course of aggregation that is largely independent of aggregate properties. In addition, the combination of these data with ThT measurements of the same reaction time points reveals important aspects of average aggregate structure at each time point. Examples of the use and potential value of AWN-ThT measurements during amyloid assembly Aβ and polyglutamine peptides are provided.

Key words

Amyloid Oligomer Thioflavin T Centrifugation HPLC Assembly intermediates Polymorphic Fibrils Nucleation Elongation 

Notes

Acknowledgment

We acknowledge financial support from N.I.H. grant R01 GM099718.

References

  1. 1.
    Wetzel R (1994) Mutations and off-pathway aggregation of proteins. Trends Biotechnol 12(5):193–198.  https://doi.org/10.1016/0167-7799(94)90082-5 CrossRefPubMedGoogle Scholar
  2. 2.
    Tyedmers J, Mogk A, Bukau B (2010) Cellular strategies for controlling protein aggregation. Nat Rev Mol Cell Biol 11(11):777–788.  https://doi.org/10.1038/nrm2993 CrossRefPubMedGoogle Scholar
  3. 3.
    Buxbaum JN, Linke RP (2012) A molecular history of the amyloidoses. J Mol Biol 421(2–3):142–159.  https://doi.org/10.1016/j.jmb.2012.01.024 CrossRefPubMedGoogle Scholar
  4. 4.
    Krieg E, Bastings MMC, Besenius P, Rybtchinski B (2016) Supramolecular polymers in aqueous media. Chem Rev 116(4):2414–2477.  https://doi.org/10.1021/acs.chemrev.5b00369 CrossRefPubMedGoogle Scholar
  5. 5.
    Chick H, Martin CJ (1912) On the “heat coagulation” of proteins. Part IV. The conditions controlling the agglutination of proteins already acted on by hot water. J Physiol 45:261–295CrossRefGoogle Scholar
  6. 6.
    Mirsky AE, Pauling L (1936) On the structure of native, denatured and coagulated protein. Proc Natl Acad Sci U S A 22:439–447CrossRefGoogle Scholar
  7. 7.
    Glenner GG, Terry W, Harada M, Isersky C, Page D (1971) Amyloid fibril proteins: proof of homology with immunoglobulin light chains by sequence analyses. Science 172(988):1150–1151CrossRefGoogle Scholar
  8. 8.
    Eanes ED, Glenner GG (1968) X-ray diffraction studies on amyloid filaments. J Histochem Cytochem 16(11):673–677CrossRefGoogle Scholar
  9. 9.
    Cohen AS, Calkins E (1959) Electron microscopic observations on a fibrous component in amyloid of diverse origins. Nature 183:1202–1203CrossRefGoogle Scholar
  10. 10.
    London J, Skrzynia C, Goldberg ME (1974) Renaturation of Escherichia coli tryptophanase after exposure to 8 M urea. Evidence for the existence of nucleation centers. Eur J Biochem 47(2):409–415CrossRefGoogle Scholar
  11. 11.
    Helms LR, Wetzel R (1996) Specificity of abnormal assembly in immunoglobulin light chain deposition disease and amyloidosis. J Mol Biol 257(1):77–86.  https://doi.org/10.1006/jmbi.1996.0148 CrossRefPubMedGoogle Scholar
  12. 12.
    Wood SJ, Maleeff B, Hart T, Wetzel R (1996) Physical, morphological and functional differences between ph 5.8 and 7.4 aggregates of the Alzheimer’s amyloid peptide Abeta. J Mol Biol 256(5):870–877.  https://doi.org/10.1006/jmbi.1996.0133 CrossRefPubMedGoogle Scholar
  13. 13.
    Walsh DM, Lomakin A, Benedek GB, Condron MM, Teplow DB (1997) Amyloid beta-protein fibrillogenesis. Detection of a protofibrillar intermediate. J Biol Chem 272(35):22364–22372CrossRefGoogle Scholar
  14. 14.
    Harper JD, Wong SS, Lieber CM, Lansbury PT (1997) Observation of metastable Abeta amyloid protofibrils by atomic force microscopy. Chem Biol 4(2):119–125CrossRefGoogle Scholar
  15. 15.
    Chemuru S, Kodali R, Wetzel R (2016) C-terminal threonine reduces Ab43 amyloidogenicity compared with Ab42. J Mol Biol 428(2 Pt A):274–291.  https://doi.org/10.1016/j.jmb.2015.06.008 CrossRefPubMedGoogle Scholar
  16. 16.
    Goldsbury CS, Cooper GJ, Goldie KN, Muller SA, Saafi EL, Gruijters WT, Misur MP, Engel A, Aebi U, Kistler J (1997) Polymorphic fibrillar assembly of human amylin. J Struct Biol 119(1):17–27CrossRefGoogle Scholar
  17. 17.
    Petkova AT, Leapman RD, Guo Z, Yau WM, Mattson MP, Tycko R (2005) Self-propagating, molecular-level polymorphism in Alzheimer’s b-amyloid fibrils. Science 307(5707):262–265CrossRefGoogle Scholar
  18. 18.
    Kodali R, Williams AD, Chemuru S, Wetzel R (2010) Abeta(1-40) forms five distinct amyloid structures whose beta-sheet contents and fibril stabilities are correlated. J Mol Biol 401(3):503–517.  https://doi.org/10.1016/j.jmb.2010.06.023 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Naiki H, Higuchi K, Hosokawa M, Takeda T (1989) Fluorometric determination of amyloid fibrils in vitro using the fluorscent dye, thioflavine T. Anal Biochem 177:244–249CrossRefGoogle Scholar
  20. 20.
    Klunk WE, Engler H, Nordberg A, Wang Y, Blomqvist G, Holt DP, Bergstrom M, Savitcheva I, Huang GF, Estrada S, Ausen B, Debnath ML, Barletta J, Price JC, Sandell J, Lopresti BJ, Wall A, Koivisto P, Antoni G, Mathis CA, Langstrom B (2004) Imaging brain amyloid in Alzheimer’s disease with Pittsburgh compound-B. Ann Neurol 55(3):306–319CrossRefGoogle Scholar
  21. 21.
    Naiki H, Higuchi K, Nakakuki K, Takeda T (1991) Kinetic analysis of amyloid fibril polymerization in vitro. Lab Investig 65(1):104–110PubMedGoogle Scholar
  22. 22.
    Collins SR, Douglass A, Vale RD, Weissman JS (2004) Mechanism of prion propagation: amyloid growth occurs by monomer addition. PLoS Biol 2(10):e321CrossRefGoogle Scholar
  23. 23.
    O’Nuallain B, Shivaprasad S, Kheterpal I, Wetzel R (2005) Thermodynamics of abeta(1-40) amyloid fibril elongation. Biochemist 44(38):12709–12718CrossRefGoogle Scholar
  24. 24.
    Chen S, Berthelier V, Hamilton JB, O’Nuallain B, Wetzel R (2002) Amyloid-like features of polyglutamine aggregates and their assembly kinetics. Biochemist 41(23):7391–7399CrossRefGoogle Scholar
  25. 25.
    O’Nuallain B, Thakur AK, Williams AD, Bhattacharyya AM, Chen S, Thiagarajan G, Wetzel R (2006) Kinetics and thermodynamics of amyloid assembly using a high-performance liquid chromatography-based sedimentation assay. Methods Enzymol 413:34–74.  https://doi.org/10.1016/S0076-6879(06)13003-7 CrossRefPubMedGoogle Scholar
  26. 26.
    Biancalana M, Koide S (2010) Molecular mechanism of Thioflavin-T binding to amyloid fibrils. Biochim Biophys Acta 1804(7):1405–1412CrossRefGoogle Scholar
  27. 27.
    LeVine IIIH (1993) Thioflavine T interaction with synthetic Alzheimer’s disease b-amyloid peptides: detection of amyloid aggregation in solution. Protein Sci 2:404–410CrossRefGoogle Scholar
  28. 28.
    Chen S, Ferrone F, Wetzel R (2002) Huntington’s disease age-of-onset linked to polyglutamine aggregation nucleation. Proc Natl Acad Sci U S A 99:11884–11889CrossRefGoogle Scholar
  29. 29.
    Misra P, Kodali R, Chemuru S, Kar K, Wetzel R (2016) Rapid α-oligomer formation mediated by the Aβ C-terminus initiates an amyloid assembly pathway. Nat Commun 7:12419CrossRefGoogle Scholar
  30. 30.
    Jan A, Hartley DM, Lashuel HA (2010) Preparation and characterization of toxic Abeta aggregates for structural and functional studies in Alzheimer’s disease research. Nat Protoc 5(6):1186–1209.  https://doi.org/10.1038/nprot.2010.72 CrossRefPubMedGoogle Scholar
  31. 31.
    Kuipers BJ, Gruppen H (2007) Prediction of molar extinction coefficients of proteins and peptides using UV absorption of the constituent amino acids at 214 nm to enable quantitative reverse phase high-performance liquid chromatography-mass spectrometry analysis. J Agric Food Chem 55(14):545–551CrossRefGoogle Scholar
  32. 32.
    Jayaraman M, Kodali R, Sahoo B, Thakur AK, Mayasundari A, Mishra R, Peterson CB, Wetzel R (2012) Slow amyloid nucleation via alpha-helix-rich oligomeric intermediates in short polyglutamine-containing huntingtin fragments. J Mol Biol 415(5):881–899.  https://doi.org/10.1016/j.jmb.2011.12.010 CrossRefPubMedGoogle Scholar
  33. 33.
    Ferrone F (1999) Analysis of protein aggregation kinetics. Methods Enzymol 309:256–274CrossRefGoogle Scholar
  34. 34.
    Knowles TP, Waudby CA, Devlin GL, Cohen SI, Aguzzi A, Vendruscolo M, Terentjev EM, Welland ME, Dobson CM (2009) An analytical solution to the kinetics of breakable filament assembly. Science 326(5959):1533–1537.  https://doi.org/10.1126/science.1178250 CrossRefPubMedGoogle Scholar
  35. 35.
    Mok YF, Howlett GJ (2006) Sedimentation velocity analysis of amyloid oligomers and fibrils. Methods Enzymol 413:199–217.  https://doi.org/10.1016/S0076-6879(06)13011-6 CrossRefPubMedGoogle Scholar
  36. 36.
    Sahoo B, Arduini I, Drombosky KW, Kodali R, Sanders LH, Greenamyre JT, Wetzel R (2016) Folding landscape of mutant Huntingtin exon1:diffusible multimers, oligomers and fibrils, and no detectable monomer. PLoS One.  https://doi.org/10.1371/journal.pone.0155747
  37. 37.
    Williams AD, Sega M, Chen M, Kheterpal I, Geva M, Berthelier V, Kaleta DT, Cook KD, Wetzel R (2005) Structural properties of Abeta protofibrils stabilized by a small molecule. Proc Natl Acad Sci U S A 102(20):7115–7120CrossRefGoogle Scholar
  38. 38.
    Kar K, Jayaraman M, Sahoo B, Kodali R, Wetzel R (2011) Critical nucleus size for disease-related polyglutamine aggregation is repeat-length dependent. Nat Struct Mol Biol 18(3):328–336.  https://doi.org/10.1038/nsmb.1992 CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Wetzel R (2006) Nucleation of huntingtin aggregation in cells. Nat Chem Biol 2(6):297–298CrossRefGoogle Scholar
  40. 40.
    Bhattacharyya AM, Thakur AK, Wetzel R (2005) Polyglutamine aggregation nucleation: thermodynamics of a highly unfavorable protein folding reaction. Proc Natl Acad Sci U S A 102(43):15400–15405CrossRefGoogle Scholar
  41. 41.
    Morimoto A, Irie K, Murakami K, Masuda Y, Ohigashi H, Nagao M, Fukuda H, Shimizu T, Shirasawa T (2004) Analysis of the secondary structure of beta-amyloid (Abeta42) fibrils by systematic proline replacement. J Biol Chem 279(50):52781–52788CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Ronald Wetzel
    • 1
  • Saketh Chemuru
    • 1
    • 2
  • Pinaki Misra
    • 1
    • 3
  • Ravi Kodali
    • 1
    • 4
  • Smita Mukherjee
    • 1
    • 5
  • Karunakar Kar
    • 1
    • 6
  1. 1.Department of Structural Biology and Pittsburgh Institute for Neurodegenerative DiseasesUniversity of Pittsburgh School of MedicinePittsburghUSA
  2. 2.Department of Physiology and BiophysicsCase Western Reserve UniversityClevelandUSA
  3. 3.Department of Biochemistry and Molecular BiologyMayo Clinic College of MedicineRochesterUSA
  4. 4.Department of Chemistry and BiochemistryDuquesne UniversityPittsburghUSA
  5. 5.The Wharton SchoolUniversity of PennsylvaniaPhiladelphiaUSA
  6. 6.School of Life SciencesJawaharlal Nehru UniversityNew DelhiIndia

Personalised recommendations