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Bioorthogonal Click Chemistry Enables Site‐specific Fluorescence Labeling of Functional NMDA Receptors for Super‐Resolution Imaging
Author(s) -
Neubert Franziska,
Beliu Gerti,
Terpitz Ulrich,
Werner Christian,
Geis Christian,
Sauer Markus,
Doose Sören
Publication year - 2018
Publication title -
angewandte chemie
Language(s) - English
Resource type - Journals
eISSN - 1521-3757
pISSN - 0044-8249
DOI - 10.1002/ange.201808951
Subject(s) - bioorthogonal chemistry , click chemistry , chemistry , fluorescence microscope , fluorescence , biophysics , fluorescence lifetime imaging microscopy , super resolution microscopy , combinatorial chemistry , biology , physics , quantum mechanics
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.