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  • Monitoring Allosteric Interactions with CXCR4 Using NanoBiT Conjugated Nanobodies.

Monitoring Allosteric Interactions with CXCR4 Using NanoBiT Conjugated Nanobodies.

Cell chemical biology (2020-07-02)
Mark Soave, Raimond Heukers, Barrie Kellam, Jeanette Woolard, Martine J Smit, Stephen J Briddon, Stephen J Hill
ABSTRACT

Camelid single-domain antibody fragments (nanobodies) offer the specificity of an antibody in a single 15-kDa immunoglobulin domain. Their small size allows for easy genetic manipulation of the nanobody sequence to incorporate protein tags, facilitating their use as biochemical probes. The nanobody VUN400, which recognizes the second extracellular loop of the human CXCR4 chemokine receptor, was used as a probe to monitor specific CXCR4 conformations. VUN400 was fused via its C terminus to the 11-amino-acid HiBiT tag (VUN400-HiBiT) which complements LgBiT protein, forming a full-length functional NanoLuc luciferase. Here, complemented luminescence was used to detect VUN400-HiBiT binding to CXCR4 receptors expressed in living HEK293 cells. VUN400-HiBiT binding to CXCR4 could be prevented by orthosteric and allosteric ligands, allowing VUN400-HiBiT to be used as a probe to detect allosteric interactions with CXCR4. These data demonstrate that the high specificity offered by extracellular targeted nanobodies can be utilized to probe receptor pharmacology.

MATERIALS
Product Number
Brand
Product Description

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