Angewandte Chemie - International Edition
Angewandte Chemie - International Edition
5 years ago

Ultrasensitive Measurement of Ca2+ Influx into Lipid Vesicles Induced by Protein Aggregates

Ultrasensitive Measurement of Ca2+ Influx into Lipid Vesicles Induced by Protein Aggregates
Christopher M. Dobson, Patrick Flagmeier, Sonia Gandhi, David Klenerman, Steven F. Lee, Tuomas P. J. Knowles, David C. Wirthensohn, Cécile Vincke, Suman De, Serge Muyldermans
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. Disruptive influence: To quantify and characterize the membrane-disrupting protein aggregates, associated with neurodegenerative diseases, a high-throughput assay based on measuring the amount of aggregate-induced Ca2+ entry into immobilized lipid vesicles has been developed. Picomolar concentrations of Aβ42 oligomers induce Ca2+ influx which can be inhibited by the addition of a chaperone and a nanobody.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/anie.201700966

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