An Aggregate Weight-Normalized Thioflavin-T Measurement Scale for Characterizing Polymorphic Amyloids and Assembly Intermediates
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 wordsAmyloid Oligomer Thioflavin T Centrifugation HPLC Assembly intermediates Polymorphic Fibrils Nucleation Elongation
We acknowledge financial support from N.I.H. grant R01 GM099718.
- 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
- 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
- 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.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
- 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