Jiang, BoUmezaki, UtanaAugustine, AndreaJayasinghe-Arachchige, Vindi M.Serafim, Leonardo F.He, Zhi Mei SoniaWyss, Kevin M.Prabhakar, RajeevMartí, Angel A.2023-02-232023-02-232023Jiang, Bo, Umezaki, Utana, Augustine, Andrea, et al.. "Deconvoluting binding sites in amyloid nanofibrils using time-resolved spectroscopy." <i>Chemical Science,</i> 14, (2023) Royal Society of Chemisty: 1072-1081. https://doi.org/10.1039/D2SC05418C.https://hdl.handle.net/1911/114475Steady-state fluorescence spectroscopy has a central role not only for sensing applications, but also in biophysics and imaging. Light switching probes, such as ruthenium dipyridophenazine complexes, have been used to study complex systems such as DNA, RNA, and amyloid fibrils. Nonetheless, steady-state spectroscopy is limited in the kind of information it can provide. In this paper, we use time-resolved spectroscopy for studying binding interactions between amyloid-β fibrillar structures and photoluminescent ligands. Using time-resolved spectroscopy, we demonstrate that ruthenium complexes with a pyrazino phenanthroline derivative can bind to two distinct binding sites on the surface of fibrillar amyloid-β, in contrast with previous studies using steady-state photoluminescence spectroscopy, which only identified one binding site for similar compounds. The second elusive binding site is revealed when deconvoluting the signals from the time-resolved decay traces, allowing the determination of dissociation constants of 3 and 2.2 μM. Molecular dynamic simulations agree with two binding sites on the surface of amyloid-β fibrils. Time-resolved spectroscopy was also used to monitor the aggregation of amyloid-β in real-time. In addition, we show that common polypyridine complexes can bind to amyloid-β also at two different binding sites. Information on how molecules bind to amyloid proteins is important to understand their toxicity and to design potential drugs that bind and quench their deleterious effects. The additional information contained in time-resolved spectroscopy provides a powerful tool not only for studying excited state dynamics but also for sensing and revealing important information about the system including hidden binding sites.engThis Open Access Article is licensed under a Creative Commons Attribution 3.0 Unported LicenceJournal articled2sc05418chttps://doi.org/10.1039/D2SC05418C