Real-time probing of β-amyloid self-assembly and inhibition using fluorescence self-quenching between neighbouring dyes

Quinn, S. D.. , Dalgarno, P. A., Cameron, R. T., Hedley, G. J. , Hacker, C., Lucocq, J. M., Baillie, G. S. , Samuel, I. D.W. and Penedo, J. C. (2014) Real-time probing of β-amyloid self-assembly and inhibition using fluorescence self-quenching between neighbouring dyes. Molecular BioSystems, 10(1), p. 34. (doi:10.1039/c3mb70272c) (PMID:24170094)

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Abstract

The fluorescence response of the Thioflavin-T (ThT) dye and derivatives has become the standard tool for detecting β-amyloid aggregates (Aβ) in solution. However, it is accepted that ThT-based methods suffer from important drawbacks. Some of these are due to the cationic structure of ThT, which limits its application at slightly acidic conditions; whereas some limitations are related to the general use of an extrinsic-dye sensing strategy and its intrinsic requirement for the formation of a sensor-binding site during the aggregation process. Here, we introduce fluorescence-self-quenching (FSQ) between N-terminally tagged peptides as a strategy to overcome some of these limitations. Using a combination of steady-state, picosecond time-resolved fluorescence and transmission electron microscopy, we characterize the fluorescence response of HiLyte fluor 555-labelled Aβ peptides and demonstrate that Aβ self-assembly organizes the covalently attached probes in close proximity to trigger the self-quenching sensing process over a broad range of conditions. Importantly, we prove that N-terminal tagging of β-amyloid peptides does not alter the self-assembly kinetics or the resulting aggregated structures. We also tested the ability of FSQ-based methods to monitor the inhibition of Aβ1–42 aggregation using the small heat-shock protein Hsp20 as a model system. Overall, FSQ-based strategies for amyloid-sensing fill the gap between current morphology-specific protocols using extrinsic dyes, and highly-specialized single-molecule techniques that are difficult to implement in high-throughput analytical determinations. When performed in Förster resonance energy transfer (FRET) format, the method becomes a ratiometric platform to gain insights into amyloid structure and for standardizing in vitro studies of amyloid aggregation.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Quinn, Dr Steven and Hedley, Dr Gordon and Baillie, Professor George and Cameron, Mr Ryan
Authors: Quinn, S. D.., Dalgarno, P. A., Cameron, R. T., Hedley, G. J., Hacker, C., Lucocq, J. M., Baillie, G. S., Samuel, I. D.W., and Penedo, J. C.
College/School:College of Medical Veterinary and Life Sciences > Institute of Cardiovascular and Medical Sciences
College of Science and Engineering > School of Chemistry
Journal Name:Molecular BioSystems
Publisher:Royal Society of Chemistry
ISSN:1742-206X
ISSN (Online):1742-2051

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