The main motivation of this work was to address the challenge of single-molecule functional study of membrane proteins under stable and independently controlled electrical and chemical membrane potentials. Although transmembrane potential is often essential for the function of membrane proteins, current in vitro systems provide only limited options for studying them under biologically relevant conditions. Our experimental assay is based on the droplet-on-hydrogel bilayer technique (Leptihn et al. Nat Protoc 8:1048–1057, 2013), where a lipid bilayer forms between a sub-millimetre water droplet and a thin hydrogel layer on a glass cover slip, enabling high-resolution microscopy in total internal reflection mode. To extend the application of this assay beyond channels to other membrane proteins, we introduce a custom-built, electronically controlled perfusion system that is designed to directly connect to the droplet above the lipid bilayer. This system can supply a stable voltage to the bilayer and is suitable for delivery of fragile membrane proteins embedded in proteoliposomes via charged fusion (Ishmukhametov et al. Nat Commun 7:13025, 2016), introducing changes of chemical potentials, and timed introduction of labels or substrate into the droplet. This work represents one of the steps towards single-molecule functional study of F1Fo ATP synthase under variable transmembrane potentials. High-resolution single-molecule observation of its rotation steps on the microsecond timescale could provide valuable insights into the mechanisms of energy transport across the molecule.
