Protocol for sample preparation and single-molecule optical tweezers study of nanodisc-embedded ABC transporter OpuA
Summary: 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 des...
Saved in:
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2025-06-01
|
| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166725002758 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850235040473022464 |
|---|---|
| author | Lyan van der Sleen Marco van den Noort Laura-Marie Silbermann Bert Poolman Katarzyna Tych |
| author_facet | Lyan van der Sleen Marco van den Noort Laura-Marie Silbermann Bert Poolman Katarzyna Tych |
| author_sort | Lyan van der Sleen |
| collection | DOAJ |
| description | Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| format | Article |
| id | doaj-art-b6cc0030ddfe42acb9ca2b8b54b72185 |
| institution | OA Journals |
| issn | 2666-1667 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj-art-b6cc0030ddfe42acb9ca2b8b54b721852025-08-20T02:02:25ZengElsevierSTAR Protocols2666-16672025-06-016210386910.1016/j.xpro.2025.103869Protocol for sample preparation and single-molecule optical tweezers study of nanodisc-embedded ABC transporter OpuALyan van der Sleen0Marco van den Noort1Laura-Marie Silbermann2Bert Poolman3Katarzyna Tych4Department of Biochemistry Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 3, 9747 AG Groningen, the Netherlands; Corresponding authorChemical Biology Group, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG Groningen, the NetherlandsChemical Biology Group, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG Groningen, the NetherlandsDepartment of Biochemistry Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 3, 9747 AG Groningen, the NetherlandsChemical Biology Group, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG Groningen, the Netherlands; Corresponding authorSummary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.http://www.sciencedirect.com/science/article/pii/S2666166725002758BiophysicsSingle-molecule assaysProtein biochemistry |
| spellingShingle | Lyan van der Sleen Marco van den Noort Laura-Marie Silbermann Bert Poolman Katarzyna Tych Protocol for sample preparation and single-molecule optical tweezers study of nanodisc-embedded ABC transporter OpuA STAR Protocols Biophysics Single-molecule assays Protein biochemistry |
| title | Protocol for sample preparation and single-molecule optical tweezers study of nanodisc-embedded ABC transporter OpuA |
| title_full | Protocol for sample preparation and single-molecule optical tweezers study of nanodisc-embedded ABC transporter OpuA |
| title_fullStr | Protocol for sample preparation and single-molecule optical tweezers study of nanodisc-embedded ABC transporter OpuA |
| title_full_unstemmed | Protocol for sample preparation and single-molecule optical tweezers study of nanodisc-embedded ABC transporter OpuA |
| title_short | Protocol for sample preparation and single-molecule optical tweezers study of nanodisc-embedded ABC transporter OpuA |
| title_sort | protocol for sample preparation and single molecule optical tweezers study of nanodisc embedded abc transporter opua |
| topic | Biophysics Single-molecule assays Protein biochemistry |
| url | http://www.sciencedirect.com/science/article/pii/S2666166725002758 |
| work_keys_str_mv | AT lyanvandersleen protocolforsamplepreparationandsinglemoleculeopticaltweezersstudyofnanodiscembeddedabctransporteropua AT marcovandennoort protocolforsamplepreparationandsinglemoleculeopticaltweezersstudyofnanodiscembeddedabctransporteropua AT lauramariesilbermann protocolforsamplepreparationandsinglemoleculeopticaltweezersstudyofnanodiscembeddedabctransporteropua AT bertpoolman protocolforsamplepreparationandsinglemoleculeopticaltweezersstudyofnanodiscembeddedabctransporteropua AT katarzynatych protocolforsamplepreparationandsinglemoleculeopticaltweezersstudyofnanodiscembeddedabctransporteropua |