NanoB2 to monitor interactions of ligands with membrane proteins by combining nanobodies and NanoBRET

van den Bor, Jelle; Bergkamp, Nick D.; Anbuhl, Stephanie M.; Dekker, Françoise; Comez, Dehan; Perez Almeria, Claudia V.; Bosma, Reggie; White, Carl W.; Kilpatrick, Laura E.; Hill, Stephen J.; Siderius, Marco; Smit, Martine J.; Heukers, Raimond

Abstract

The therapeutic potential of ligands targeting disease-associated membrane proteins is predicted by ligand-receptor binding constants, which can be determined using NanoLuciferase (NanoLuc)-based bioluminescence resonance energy transfer (NanoBRET) methods. However, the broad applicability of these methods is hampered by the restricted availability of fluorescent probes. We describe the use of antibody fragments, like nanobodies, as universal building blocks for fluorescent probes for use in NanoBRET. Our nanobody-NanoBRET (NanoB2) workflow starts with the generation of NanoLuc-tagged receptors and fluorescent nanobodies, enabling homogeneous, real-time monitoring of nanobody-receptor binding. Moreover, NanoB2 facilitates the assessment of receptor binding of unlabeled ligands in competition binding experiments. The broad significance is illustrated by the successful application of NanoB2 to different drug targets (e.g., multiple G protein-coupled receptors [GPCRs] and a receptor tyrosine kinase [RTK]) at distinct therapeutically relevant binding sites (i.e., extracellular and intracellular).

Citation

van den Bor, J., Bergkamp, N. D., Anbuhl, S. M., Dekker, F., Comez, D., Perez Almeria, C. V., Bosma, R., White, C. W., Kilpatrick, L. E., Hill, S. J., Siderius, M., Smit, M. J., & Heukers, R. (2023). NanoB2 to monitor interactions of ligands with membrane proteins by combining nanobodies and NanoBRET. Cell Reports Methods, 3(3), Article 100422. https://doi.org/10.1016/j.crmeth.2023.100422

• Journal Article Type: Article
• Acceptance Date: Feb 17, 2023
• Online Publication Date: Mar 13, 2023
• Publication Date: Mar 27, 2023
• Deposit Date: Mar 30, 2023
• Publicly Available Date: Apr 4, 2023
• Journal: Cell Reports Methods
• Electronic ISSN: 2667-2375
• Publisher: Elsevier (Cell Press)
• Peer Reviewed: Peer Reviewed
• Volume: 3
• Issue: 3
• Article Number: 100422
• DOI: https://doi.org/10.1016/j.crmeth.2023.100422
• Public URL: https://nottingham-repository.worktribe.com/output/19006199
• Publisher URL: https://www.sciencedirect.com/science/article/pii/S2667237523000334?via%3Dihub