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

Lyan van der Sleen, Marco van den Noort, Laura-Marie Silbermann, Bert Poolman, Katarzyna Tych

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Article in STAR protocols, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.

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5 authors.

Lyan van der SleenDepartment of Biochemistry Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 3, 9747 AG Groningen, the Netherlands. Electronic address: l.m.van.der.sleen@rug.nl.

Marco van den NoortChemical Biology Group, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG Groningen, the Netherlands.

Laura-Marie SilbermannChemical Biology Group, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG Groningen, the Netherlands.

Bert PoolmanDepartment of Biochemistry Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 3, 9747 AG Groningen, the Netherlands.

Katarzyna TychChemical Biology Group, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG Groningen, the Netherlands. Electronic address: k.m.tych@rug.nl.

Funding

No grant is acknowledged in the PubMed record.

6 · The paper itself

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.

Indexed as

ATP-Binding Cassette TransportersEscherichia coli ProteinsNanostructuresOptical TweezersSingle Molecule ImagingATP-Binding Cassette TransportersEscherichia coli ProteinsBiophysicsProtein biochemistrySingle-molecule assays

Identifiers

PMID40460202

DOI10.1016/j.xpro.2025.103869

PMCPMC12167795

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