Probing supramolecular protein assembly using covalently attached fluorescent molecular rotors

Abstract

Changes in microscopic viscosity and macromolecular crowding accompany the transition of proteins from their monomeric forms into highly organised fibrillar states. Previously, we have demonstrated that viscosity sensitive fluorophores termed 'molecular rotors', when freely mixed with monomers of interest, are able to report on changes in microrheology accompanying amyloid formation, and measured an increase in rigidity of approximately three orders of magnitude during aggregation of lysozyme and insulin. Here we extend this strategy by covalently attaching molecular rotors to several proteins capable of assembly into fibrils, namely lysozyme, fibrinogen and amyloid-β peptide (Aβ(1-42)). We demonstrate that upon covalent attachment the molecular rotors can successfully probe supramolecular assembly in vitro. Importantly, our new strategy has wider applications in cellulo and in vivo, since covalently attached molecular rotors can be successfully delivered in situ and will colocalise with the aggregating protein, for example inside live cells. This important advantage allowed us to follow the microscopic viscosity changes accompanying blood clotting and during Aβ(1-42) aggregation in live SH-SY5Y cells. Our results demonstrate that covalently attached molecular rotors are a widely applicable tool to study supramolecular protein assembly and can reveal microrheological features of aggregating protein systems both in vitro and in cellulo not observable through classical fluorescent probes operating in light switch mode.

Molecular rotors report on changes in live cell plasma membrane microviscosity upon interaction with beta-amyloid aggregates

“…At the same time we have also used molecular rotors to monitor the changes in crowding during the aggregation of Aβ in the presence of live cells. 40 We found that beta-amyloid accumulates on the plasma membranes and adjacent to them, possibly contributing to the amyloid-induced cytotoxicity. We have also established, by monitoring the fluorescence lifetime of an Aβ-bound molecular rotor, that Aβ aggregation in cellulo proceeds via a different pathway, compared to the in vitro scenario in the absence of cells.…”

“…To investigate the effect of Aβ(1-42) oligomers on vesicles and plasma membranes, Aβ(1-42), oligomers were prepared according to the published protocol 40 ; Thioflavin T intensity assay 72 was used to confirm the correct course of Aβ(1-42) aggregation (Fig. S6).…”

“…We have also established, by monitoring the fluorescence lifetime of an Aβ-bound molecular rotor, that Aβ aggregation in cellulo proceeds via a different pathway, compared to the in vitro scenario in the absence of cells. 40 To the best of our knowledge, despite the perceived importance of Aβ-plasma membrane interactions, no direct data exist on membrane microviscosity of live cells in the presence of Aβ aggregates. Hence, in this work we use membrane-localised molecular rotor BODIPY 1 37 to investigate the effect of the interaction of aggregating beta-amyloid with plasma membranes on the plasma membrane microviscosity and organisation, in a dynamic and quantitative manner.…”

Probing biotin receptors in cancer cells with rationally designed fluorogenic squaraine dimers

“…23,24 Second, molecular rotors, 25 which are poorly uorescent in aqueous media, but light up upon binding to the targets with highly viscous environment. 26,27 One should also mention aggregation-induced emission approach, where the nonemissive dyes light up because the target triggers dye aggregation. 28 Of particular interest are probes operating by aggregation-caused quenching (ACQ) mechanism.…”

Rapid Discovery of Illuminating Peptides for Instant Detection of Opioids in Blood and Body Fluids

“…The fluorescent-activation property of MG makes it an ideal candidate for the development of illuminating peptide-dye conjugates, which enables the dynamic detection of target–ligand interactions in the near infra-red range, whilst minimizing the background autofluorescence. Covalently attached molecular rotors are a widely applicable tool to study supramolecular protein assembly, and they can reveal the microrheological features of aggregating protein systems both in vitro and in cellulo, which are not otherwise observable through classical fluorescent probes operating in the light switch mode [24]. However, it is important to emphasize that the direct conjugation of MRDs to known protein ligands has not been well explored.…”