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

## TL;DR

This paper provides a detailed protocol for studying the unfolding and interdomain interactions of the OpuA transporter using single-molecule optical tweezers.

## Contribution

A new protocol is introduced for studying membrane protein unfolding and interactions using single-molecule optical tweezers.

## Key findings

- The protocol includes steps for protein expression, purification, and lipid nanodisc reconstitution.
- It offers guidance for measuring unfolding and interdomain interactions using optical tweezers.
- The method can be applied to study membrane proteins in general.

## Abstract

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

•Instructions for sample preparation of a membrane protein for optical tweezers•Guidance on measuring unfolding and interdomain interactions using optical tweezers•Steps to analyze and interpret optical tweezers data on unfolding and interactions

Instructions for sample preparation of a membrane protein for optical tweezers

Guidance on measuring unfolding and interdomain interactions using optical tweezers

Steps to analyze and interpret optical tweezers data on unfolding and interactions

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

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.