Protocol for sample preparation and single-molecule optical tweezers study of nanodisc-embedded ABC transporter OpuA

OpuA is an osmoregulatory ATP-binding cassette transporter that undergoes different conformations upon varying salt concentrations and ligand conditions. Here, we present a protocol to study unfolding and interdomain interactions of OpuA using single-molecule optical tweezers (smOT). We describe steps for expression and purification of the protein, lipid nanodisc reconstitution, and sample preparation. We then detail procedures for smOT experiments and data analysis. This protocol also has potential application in the study of interdomain interactions and unfolding of (membrane) proteins in general. For complete details on the use and execution of this protocol, please refer to van der Sleen et al.¹.