Tunable Electromechanical Nanopore Trap Reveals Populations of Peripheral Membrane Protein Binding Conformations

We demonstrate that a naturally occurring nanopore, the voltage-dependent anion channel (VDAC) of the mitochondrion, can be used to electromechanically trap and interrogate proteins bound to a lipid surface at the single-molecule level. Electromechanically probing α-synuclein (αSyn), an intrinsically disordered neuronal protein intimately associated with Parkinson's pathology, reveals wide variation in the time required for individual proteins to unbind from the same membrane surface. The observed distributions of unbinding times span up to 3 orders of magnitude and depend strongly on the lipid composition of the membrane; surprisingly, lipid membranes to which αSyn binds weakly are most likely to contain subpopulations in which electromechanically driven unbinding is very slow. We conclude that unbinding of αSyn from the membrane surface depends not only on membrane binding affinity but also on the conformation adopted by an individual αSyn molecule on the membrane surface.