Calcein Release Assay to Measure Membrane Permeabilization by Recombinant Alpha-Synuclein

Abstract

Lipid membranes are involved in regulating biochemical and biological processes and in modulating the selective permeability of cells, organelles, and vesicles. Membrane composition, charge, curvature, and fluidity all have concerted effects on cellular signaling and homeostasis. The ability to prepare artificial lipid assemblies that mimic biological membranes has enabled investigators to obtain considerable insight into biomolecule-membrane interactions. Lipid nanoscale assemblies can vary greatly in size and composition and can consist of a single lipid monolayer, a bilayer, or other more complex assemblies. This structural diversity makes liposomes suitable for a wide variety of biochemical and clinical applications. Here, we describe a calcein dye leakage assay that we have developed to monitor phospholipid vesicle disruption by alpha-synuclein (αSyn), a presynaptic protein that plays a central role in Parkinson’s disease (PD). We present data showing the effect of adenylylation of αSyn on αSyn-mediated vesicle disruption as an example. This assay can be used to study the effect of mutations or post-translational modifications on αSyn-membrane interactions, to identify protein binding partners or chemical entities that perturb these interactions, and to study the effects of different lipids on the permeabilization activity of αSyn or any other protein.