By the same authors

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

Research output: Contribution to journalArticle

Published copy (DOI)

Author(s)

  • Steven D. Quinn
  • Paul A. Dalgarno
  • Ryan T. Cameron
  • Gordon J. Hedley
  • Christian Hacker
  • John M. Lucocq
  • George S. Baillie
  • Ifor D. W. Samuel
  • J. Carlos Penedo

Department/unit(s)

Publication details

JournalMolecular BioSystems
DatePublished - Jan 2014
Issue number1
Volume10
Number of pages11
Pages (from-to)34-44
Original languageEnglish

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.

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