Probing supramolecular protein assembly using covalently attached fluorescent molecular rotors

Kubánková, Markéta, López-Duarte, Ismael, Bull, James A, Vadukul, Devkee M, Serpell, Louise C, de Saint Victor, Marie, Stride, Eleanor and Kuimova, Marina K (2017) Probing supramolecular protein assembly using covalently attached fluorescent molecular rotors. Biomaterials, 139. pp. 195-201. ISSN 0142-9612

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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.

Item Type: Article
Schools and Departments: School of Life Sciences > Biochemistry
Research Centres and Groups: Dementia Research Group
Depositing User: Louise Serpell
Date Deposited: 28 Jul 2017 08:41
Last Modified: 02 Jul 2019 19:00

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