title: Real-time probing of β-amyloid self-assembly and inhibition using fluorescence self-quenching between neighbouring dyes.
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.
authors.affiliation: 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.
authors.forename: Steven D
authors.initials: SD
authors.lastname: Quinn
authors.forename: Paul A
authors.initials: PA
authors.lastname: Dalgarno
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Cameron
authors.forename: Gordon J
authors.initials: GJ
authors.lastname: Hedley
authors.forename: Christian
authors.initials: C
authors.lastname: Hacker
authors.forename: John M
authors.initials: JM
authors.lastname: Lucocq
authors.forename: George S
authors.initials: GS
authors.lastname: Baillie
authors.forename: Ifor D W
authors.initials: ID
authors.lastname: Samuel
authors.forename: J Carlos
authors.initials: JC
authors.lastname: Penedo
mesh_terms: D016229:Amyloid beta-Peptides / Q000737:chemistry / Q000302:isolation & purification*; D052160:Benzothiazoles; D001665:Binding Sites; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification*; D011485:Protein Binding; D013844:Thiazoles / Q000737:chemistry
chemical_list: D016229:Amyloid beta-Peptides; D052160:Benzothiazoles; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D013844:Thiazoles; C009462:thioflavin T