Alternative Pathways of Human Islet Amyloid Polypeptide Aggregation Distinguished by¹⁹F Nuclear Magnetic Resonance-Detected Kinetics of Monomer Consumption

Abstract

Amyloid formation, a complex process involving many intermediate states, is proposed to be the driving force for amyloid-related toxicity in common degenerative diseases. Unfortunately, the details of this process have been obscured by the limitations in the methods that can follow this reaction in real-time. We show that alternative pathways of aggregation can be distinguished by using 19F NMR to monitor monomer consumption along with complementary measurements of fibrillogenesis. The utility of this technique is demonstrated by tracking amyloid formation in the diabetes-related islet amyloid polypeptide (IAPP). Using this technique, we show IAPP fibrillizes without an appreciable build up of non-fibrillar intermediates, in contrast to the well-studied Aβ and α-synuclein proteins. To further develop the usage of 19F NMR, we have tracked the influence of the polyphenolic amyloid inhibitor epigallocatechin gallate (EGCG) on the aggregation pathway. Polyphenols have been shown to strongly inhibit amyloid formation in many systems. However, spectroscopic measurements of amyloid inhibition by these compounds can be severely compromised by background signals and competitive binding with extrinsic probes. Using 19F NMR, we show that thioflavin T strongly competes with EGCG for binding sites on IAPP fibers. By comparing the rates of monomer consumption and fiber formation, we are able to show that EGCG stabilizes non-fibrillar large aggregates during fibrillogenesis.

Reduced Lipid Bilayer Thickness Regulates the Aggregation and Cytotoxicity of Amyloid-β

“…Similar fluorescence reduction has previously been observed in small molecule-induced disaggregation of various amyloid fibrils, suggesting that DLPC LUVs may alter the structure of the traditionally stable fiber (49 -51). Although ThT displacement is a possibility, the stabilization of the final fluorescence at ϳ40% of the original value, instead of returning to the baseline, suggests that ThT binding is maintained to some extent and that the changes are primarily structural (29). Additionally, the reduction in ThT fluorescence was independent of the concentration of ThT in solution, further supporting the change resulting from structural alterations (supplemental Fig.…”

“…When A␤ aggregated in the presence of DLPC at stoichiometric (10 M; 1:1 lipid/peptide ratio) or higher lipid concentrations, ThT fluorescence remained at baseline levels, which suggests no fibril formation. ThT will occasionally provide false positives of inhibition resulting from dye displacement or the inability of unique amyloid sequences to bind the dye, despite containing the canonical ␤-strand fold (29,30). To confirm that the reduction in ThT fluorescence was the result of structural change, CD and TEM were performed on end stage aggregates (Fig.…”

Insights into antiamyloidogenic properties of the green tea extract (−)-epigallocatechin-3-gallate toward metal-associated amyloid-β species

“…S2) by gel electrophoresis with Western blotting using an anti-Aβ antibody (6E10), as well as by transmission electron microscopy (TEM). These methods were used to visualize the changes in size distribution and morphology that are not accurately represented by other biological assays [e.g., thioflavin-T (ThT) assay] (37,38).…”

Escherichia coli Topoisomerase IV E Subunit and an Inhibitor Binding Mode Revealed by NMR Spectroscopy

“…Based on these H-D exchange experiments, residues that are critical for and affected by 2 binding were revealed, suggesting that this method will also be useful for understanding protein-drug interactions. 19 F NMR experiments are widely used to study ligand-protein binding and dynamics (28,29). The simplicity and high sensitivity (natural abundance of 19 F ϭ 100%) makes the application of such techniques attractive (30).…”