Bioorthogonal Click Chemistry Enables Site‐specific Fluorescence Labeling of Functional NMDA Receptors for Super‐Resolution Imaging. (15th November 2018)

• Record Type: Journal Article
• Authors: Neubert, Franziska; Beliu, Gerti; Terpitz, Ulrich; Werner, Christian; Geis, Christian; Sauer, Markus; Doose, Sören
• Is Part Of: Angewandte Chemie. Volume 130:Number 50(2018)
• Page Start: 16602
• Page End: 16607
• Publication Date: 2018-11-15
• Subjects: Einzelmolekül-Lokalisationsmikroskopie -- Erweiterung des genetischen Codes -- Fluoreszenzsonden -- Nichtnatürliche Aminosäuren; Chemistry -- Periodicals; 540
• DOI: 10.1002/ange.201808951
• Languages: English
• ISSNs: 0044-8249

Abstract

Abstract: Super‐resolution microscopy requires small fluorescent labels. We report the application of genetic code expansion in combination with bioorthogonal click chemistry to label the NR1 domain of the NMDA receptor. We generated NR1 mutants incorporating an unnatural amino acid at various positions in order to attach small organic fluorophores such as Cy5‐tetrazine site‐specifically to the extracellular domain of the receptor. Mutants were optimized with regard to protein expression, labeling efficiency and receptor functionality as tested by fluorescence microscopy and whole‐cell patch clamp. The results show that bioorthogonal click chemistry in combination with small organic dyes is superior to available immunocytochemistry protocols for receptor labeling in live and fixed cells and enables single‐molecule sensitive super‐resolution microscopy experiments.