Real-time probing of β-amyloid self-assembly and inhibition using fluorescence self-quenching between neighbouring dyes
| Autor: | J. Carlos Penedo, John M. Lucocq, Paul A. Dalgarno, Gordon J. Hedley, Christian Hacker, Steven D. Quinn, Ifor D. W. Samuel, George S. Baillie, Ryan T. Cameron |
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| Rok vydání: | 2014 |
| Předmět: |
Amyloid beta-Peptides
Binding Sites Quenching (fluorescence) Amyloid Chemistry Kinetics Nanotechnology Fluorescence Peptide Fragments Thiazoles Förster resonance energy transfer Covalent bond Picosecond Biophysics Humans Benzothiazoles Self-assembly Molecular Biology Fluorescent Dyes Protein Binding Biotechnology |
| Zdroj: | Mol. BioSyst.. 10:34-44 |
| ISSN: | 1742-2051 1742-206X |
| DOI: | 10.1039/c3mb70272c |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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