Genetic Code Expansion and Click-Chemistry Labeling to Visualize GABA-A Receptors by Super-Resolution Microscopy
Zitieren Sie bitte immer diese URN: urn:nbn:de:bvb:20-opus-251035
- Fluorescence labeling of difficult to access protein sites, e.g., in confined compartments, requires small fluorescent labels that can be covalently tethered at well-defined positions with high efficiency. Here, we report site-specific labeling of the extracellular domain of γ-aminobutyric acid type A (GABA-A) receptor subunits by genetic code expansion (GCE) with unnatural amino acids (ncAA) combined with bioorthogonal click-chemistry labeling with tetrazine dyes in HEK-293-T cells and primary cultured neurons. After optimization of GABA-AFluorescence labeling of difficult to access protein sites, e.g., in confined compartments, requires small fluorescent labels that can be covalently tethered at well-defined positions with high efficiency. Here, we report site-specific labeling of the extracellular domain of γ-aminobutyric acid type A (GABA-A) receptor subunits by genetic code expansion (GCE) with unnatural amino acids (ncAA) combined with bioorthogonal click-chemistry labeling with tetrazine dyes in HEK-293-T cells and primary cultured neurons. After optimization of GABA-A receptor expression and labeling efficiency, most effective variants were selected for super-resolution microscopy and functionality testing by whole-cell patch clamp. Our results show that GCE with ncAA and bioorthogonal click labeling with small tetrazine dyes represents a versatile method for highly efficient site-specific fluorescence labeling of proteins in a crowded environment, e.g., extracellular protein domains in confined compartments such as the synaptic cleft.…
Autor(en): | Alexander Kuhlemann, Gerti Beliu, Dieter Janzen, Enrica Maria Petrini, Danush Taban, Dominic A. Helmerich, Sören Doose, Martina Bruno, Andrea Barberis, Carmen Villmann, Markus Sauer, Christian Werner |
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URN: | urn:nbn:de:bvb:20-opus-251035 |
Dokumentart: | Artikel / Aufsatz in einer Zeitschrift |
Institute der Universität: | Medizinische Fakultät / Institut für Klinische Neurobiologie |
Fakultät für Biologie / Theodor-Boveri-Institut für Biowissenschaften | |
Fakultät für Biologie / Rudolf-Virchow-Zentrum | |
Sprache der Veröffentlichung: | Englisch |
Titel des übergeordneten Werkes / der Zeitschrift (Englisch): | Frontiers in Synaptic Neuroscience |
ISSN: | 1663-3563 |
Erscheinungsjahr: | 2021 |
Band / Jahrgang: | 13 |
Aufsatznummer: | 727406. |
Originalveröffentlichung / Quelle: | Frontiers in Synaptic Neuroscience (2021) 13:727406. doi: 10.3389/fnsyn.2021.727406 |
DOI: | https://doi.org/10.3389/fnsyn.2021.727406 |
Allgemeine fachliche Zuordnung (DDC-Klassifikation): | 6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit |
Freie Schlagwort(e): | GABA-A receptor; click-chemistry; dSTORM; genetic code expansion; super-resolution microscopy (SRM) |
Datum der Freischaltung: | 11.02.2022 |
Datum der Erstveröffentlichung: | 26.11.2021 |
Open-Access-Publikationsfonds / Förderzeitraum 2021 | |
Lizenz (Deutsch): | CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International |