Fast flow microfluidics and single-molecule fluorescence for the rapid characterization of α-Synuclein oligomers

Authors

Mathew H. Horrocks, University of CambridgeFollow
Laura Tosatto, University of Cambridge
Alexander J. Dear, University of Cambridge
Gonzalo A. Garcia, University of Cambridge
Marija Iljina, University of Cambridge
Nunilo Cremades, University of Cambridge
Mauro Dalla Serra, Istituto di Biofisica
Tuomas P. J Knowles, University of Cambridge
Christopher M. Dobson, University of CambridgeFollow
David Klenerman, University of Cambridge

RIS ID

104154

Publication Details

Horrocks, M. H., Tosatto, L., Dear, A. J., Garcia, G. A., Iljina, M., Cremades, N., Dalla Serra, M., Knowles, T. P. J., Dobson, C. M. & Klenerman, D. (2015). Fast flow microfluidics and single-molecule fluorescence for the rapid characterization of α-Synuclein oligomers. Analytical Chemistry, 87 (17), 8818-8826.

Abstract

α-Synuclein oligomers can be toxic to cells and may be responsible for cell death in Parkinson's disease. Their typically low abundance and highly heterogeneous nature, however, make such species challenging to study using traditional biochemical techniques. By combining fast-flow microfluidics with single-molecule fluorescence, we are able to rapidly follow the process by which oligomers of αS are formed and to characterize the species themselves. We have used the technique to show that populations of oligomers with different FRET efficiencies have varying stabilities when diluted into low ionic strength solutions. Interestingly, we have found that oligomers formed early in the aggregation pathway have electrostatic repulsions that are shielded in the high ionic strength buffer and therefore dissociate when diluted into lower ionic strength solutions. This property can be used to isolate different structural groups of αS oligomers and can help to rationalize some aspects of αS amyloid fibril formation.