NDLI: Real-time probing of ß-amyloid self-assembly and inhibition using fluorescence self-quenching between neighbouring dyes.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Quinn, Steven D. Dalgarno, Paul A. Cameron, Ryan T. Hedley, Gordon J. Hacker, Christian Lucocq, John M. Baillie, George S. Samuel, Ifor D. W. Penedo, J. Carlos
Description	Author Affiliation: Quinn SD ( SUPA School of Physics and Astronomy, University of St Andrews, North Haugh, Fife, Scotland, KY16 9SS, UK. jcp10@st-andrews.ac.uk, idws@st-andrews.ac.uk.)
Abstract	The fluorescence response of the Thioflavin-T (ThT) dye and derivatives has become the standard tool for detecting ß-amyloid aggregates (Aß) 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 Aß peptides and demonstrate that Aß 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 ß-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 Aß1-42 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.
File Format	HTM / HTML
ISSN	1742206X
Issue Number	1
Volume Number	10
e-ISSN	17422051
Journal	Molecular BioSystems
Language	English
Publisher	Royal Society of Chemistry
Publisher Date	2014-01-01
Publisher Place	Great Britain (UK)
Access Restriction	Subscribed
Subject Keyword	Discipline Molecular Biology Discipline Biochemistry Amyloid Beta-peptides Isolation & Purification Fluorescent Dyes Chemistry Peptide Fragments Binding Sites Fluorescence Humans Kinetics Protein Binding Thiazoles Journal Article
Content Type	Text
Resource Type	Article
Subject	Molecular Biology Biotechnology

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