Ultrasensitive measurement of Ca2+ influx into lipid vesicles induced by protein aggregates
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Patrick Flagmeier Suman De David C Wirthensohn Steven F Lee Cécile Vincke Serge Muyldermans Tuomas PJ Knowles Sonia Gandhi Christopher M Dobson David KlenermanAbstract
To quantify and characterize the potentially toxic protein aggregates associated with neurodegenerative diseases, a high-throughput assay based on measuring the extent of aggregate-induced Ca2+ entry into individual lipid vesicles has been developed. This approach was implemented by tethering vesicles containing a Ca2+ sensitive fluorescent dye to a passivated surface and measuring changes in the fluorescence as a result of membrane disruption using total internal reflection microscopy. Picomolar concentrations of Aβ42 oligomers could be observed to induce Ca2+ influx, which could be inhibited by the addition of a naturally occurring chaperone and a nanobody designed to bind to the Aβ peptide. We show that the assay can be used to study aggregates from other proteins, such as α-synuclein, and to probe the effects of complex biofluids, such as cerebrospinal fluid, and thus has wide applicability.
Journal details
Volume 56
Issue number 27
Pages 7750-7754
Available online
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Publisher website (DOI) 10.1002/anie.201700966
Europe PubMed Central 28474754
Pubmed 28474754
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