Single-Molecule Fluorescence Imaging of low Affinity Binding Interactions in Pacemaker ion Channels

Abstract

Molecular recognition is a cornerstone of cellular signaling processes. However, most observations of binding rely on ensemble measurements that average over and thereby obscure the individual underlying steps. Single-molecule fluorescence can reveal these dynamic events, but is traditionally limited to low concentrations of fluorophores only suitable for studying high affinity reactions. Here, we show how a combination of fluorescence resonance energy transfer (FRET) and zero-mode waveguide nanofabricated devices can be used to study single-molecule association dynamics at the micromolar to millimolar concentrations necessary for many relevant signaling pathways. As an exemplar system, we apply this approach to cyclic nucleotide binding at its receptor domain from hyperpolarization cyclic nucleotide-gated (HCN) ion channels. Our observations reveal the underlying dynamic events during cyclic nucleotide binding: an initial encounter complex when the binding site is in its receptive state, followed by an isomerization of the bound complex that traps the ligand.