Bioorthogonal Click Chemistry Enables Site-specific Fluorescence Labeling of Functional NMDA Receptors for Super-Resolution Imaging

Angew Chem Int Ed Engl. 2018 Dec 10;57(50):16364-16369. doi: 10.1002/anie.201808951. Epub 2018 Nov 15.

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.

Keywords: fluorescent probes; genetic code expansion; single-molecule localization microscopy; unnatural amino acids.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carbocyanines / chemistry*
  • Click Chemistry / methods*
  • Fluorescence
  • Fluorescent Dyes / chemistry*
  • HEK293 Cells
  • Humans
  • Microscopy, Fluorescence
  • Models, Molecular
  • Mutation
  • Optical Imaging
  • Protein Domains
  • Protein Engineering
  • Receptors, N-Methyl-D-Aspartate / analysis*
  • Receptors, N-Methyl-D-Aspartate / genetics
  • Staining and Labeling

Substances

  • Carbocyanines
  • Fluorescent Dyes
  • Receptors, N-Methyl-D-Aspartate
  • cyanine dye 5