Hongze Gang, Céline Galvagnion, Georg Meisl, Thomas Müller, Manuela Pfammatter, Alexander K. Buell, Aviad Levin, Christopher M. Dobson, Bozhong Mu and Tuomas P. J. Knowles
Anal. Chem 2018 January 9, 90 (5) 3284-3290
Elucidation of the fundamental interactions of proteins with biological membranes under native conditions is crucial for understanding the molecular basis of their biological function and malfunction. Notably, the large surface to volume ratio of living cells provides a molecular landscape for significant interactions of cellular components with membranes modulating their function. However, such interactions can be challenging to probe using conventional biophysical methods due to the heterogeneity of the species and processes involved.
Here, Gang et al use direct measurements of micron scale molecular diffusivity to detect and quantify the interactions of α-synuclein, associated with the aetiology of Parkinson’s disease, with negatively charged lipid vesicles. They further demonstrate that this microfluidic approach enables the characterisation of vesicles of different binary mixtures size distribution, which is not readily accessible using conventional light scattering techniques.
Finally, the size distributions of the two α-synuclein conformations - free α-synuclein and membrane-bound α-synuclein - were resolved under varying lipid:protein ratios, thus allowing the determination of the dissociation constant and the binding stoichiometry associated with this protein-lipid system.
The microfluidic diffusional sizing platform allows these measurements to be performed on a time scale of minutes using microlitre volumes, thus establishing the basis for an approach for the study of molecular interactions of heterogenous systems under native conditions.