Visualizing a one-way protein encounter complex by ultrafast single-molecule mixing

Abstract

A laminar flow mixing microfluidic device enables single-molecule fluorescence resonance energy transfer (FRET) kinetic measurements with a time resolution of 0.2 ms, enabling the study of early binding-coupled folding and unfolding events of an intrinsically disordered protein, α-synuclein. Also in this issue, Kim et al. describe another microfluidic mixing device for single-molecule experiments. We combined rapid microfluidic mixing with single-molecule fluorescence resonance energy transfer to study the folding kinetics of the intrinsically disordered human protein α-synuclein. The time-resolution of 0.2 ms revealed initial collapse of the unfolded protein induced by binding with lipid mimics and subsequent rapid formation of transient structures in the encounter complex. The method also enabled analysis of rapid dissociation and unfolding of weakly bound complexes triggered by massive dilution.