%0 Journal Article
%A Kubánková, Markéta
%A López Duarte, Ismael
%A Bull, James A.
%A Vadukul, Devkee M.
%A Serpell, Louise C.
%A Victor, Marie de Saint
%A Stride, Eleanor
%A Kuimova, Marina K.
%T Probing supramolecular protein assembly using covalently attached fluorescent molecular rotors
%D 2017
%@ 0142-9612
%U https://hdl.handle.net/20.500.14352/114924
%X Changes in microscopic viscosity and macromolecular crowding accompany the transition of proteinsfrom their monomeric forms into highly organised fibrillar states. Previously, we have demonstrated thatviscosity 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 anincrease in rigidity of approximately three orders of magnitude during aggregation of lysozyme andinsulin. Here we extend this strategy by covalently attaching molecular rotors to several proteins capableof assembly into fibrils, namely lysozyme, fibrinogen and amyloid-b peptide (Ab(1e42)). 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, sincecovalently attached molecular rotors can be successfully delivered in situ and will colocalise with theaggregating protein, for example inside live cells. This important advantage allowed us to follow themicroscopic viscosity changes accompanying blood clotting and during Ab(1e42) aggregation in live SHSY5Y cells. Our results demonstrate that covalently attached molecular rotors are a widely applicable toolto study supramolecular protein assembly and can reveal microrheological features of aggregatingprotein systems both in vitro and in cellulo not observable through classical fluorescent probes operatingin light switch mode.
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