Label-Free Characterization of Amyloids and Alpha-Synuclein Polymorphs by Exploiting Their Intrinsic Fluorescence Property.
Davis, Molly Jo
American Chemical Society (ACS)
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Chung, C. W., Stephens, A. D., Ward, E., Feng, Y., Davis, M. J., Kaminski, C. F., & Kaminski Schierle, G. S. (2022). Label-Free Characterization of Amyloids and Alpha-Synuclein Polymorphs by Exploiting Their Intrinsic Fluorescence Property.. Anal Chem, 94 (13), 5367-5374. https://doi.org/10.1021/acs.analchem.1c05651
Funder: Infinitus China Ltd.
Conventional in vitro aggregation assays often involve tagging with extrinsic fluorophores, which can interfere with aggregation. We propose the use of intrinsic amyloid fluorescence lifetime probed using two-photon excitation and represented by model-free phasor plots as a label-free assay to characterize the amyloid structure. Intrinsic amyloid fluorescence arises from the structured packing of β-sheets in amyloids and is independent of aromatic-based fluorescence. We show that different amyloids [i.e., α-Synuclein (αS), β-Lactoglobulin (βLG), and TasA] and different polymorphic populations of αS (induced by aggregation in salt-free and salt buffers mimicking the intra-/extracellular environments) can be differentiated by their unique fluorescence lifetimes. Moreover, we observe that disaggregation of the preformed fibrils of αS and βLG leads to increased fluorescence lifetimes, distinct from those of their fibrillar counterparts. Our assay presents a medium-throughput method for rapid classification of amyloids and their polymorphs (the latter of which recent studies have shown lead to different disease pathologies) and for testing small-molecule inhibitory compounds.
Amyloid, Fluorescence, alpha-Synuclein, Amyloidogenic Proteins, Protein Conformation, beta-Strand
Is supplemented by: https://doi.org/10.17863/CAM.82267
Medical Research Council (MR/K02292X/1)
Wellcome Trust (065807/Z/01/Z)
Wellcome Trust (203249/Z/16/Z)
Engineering and Physical Sciences Research Council (EP/S022953/1)
External DOI: https://doi.org/10.1021/acs.analchem.1c05651
This record's URL: https://www.repository.cam.ac.uk/handle/1810/336436
Attribution-NonCommercial-NoDerivatives 4.0 International
Licence URL: https://creativecommons.org/licenses/by-nc-nd/4.0/