Super-resolution microscopyrequires small fluorescent labels.Wereport the application of genetic code expansion in combination with bioorthogonal click chemistry to label the NR1 domain of the NMDAr eceptor.W eg enerated 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.M utants were optimizedw ith regardt op rotein expression, labeling efficiency and receptor functionality as tested by fluorescence microscopyand whole-cell patchclamp. The results showt hat bioorthogonal clickc hemistry in combination with small organic dyes is superior to available immunocytochemistry protocols for receptor labeling in live and fixedc ells and enables single-molecule sensitive superresolution microscopyexperiments. Super-resolutionmicroscopyprovidesspatialresolutionwellbeyond the diffraction limit on the order of af ew tens of nanometers.A st he ability to resolve structures in fluorescence microscopy not only depends on the optical resolution but also on labeling size and density,there is astrong need for efficient fluorescence labeling methods. [1] Ideally,t he fluorescent label should be small (relative to the optical resolution), specific (targeted against aw ell determined antigen), effective (with ah igh labeling efficiency), show appropriate photophysical behavior (depending on the applied imaging technique), and should not impair target function. Small organic fluorophores have the smallest footprint if incorporated by site-specific chemistry on as mall carrier. [2] Va rious approaches exist to reduce the carrier size, such as using genetically encoded tags [3] nanobodies, [4] affimers, [5] aptamers, [6] super-binding peptides, [7] or rare amino acid side chains.

γ-Aminobutyric acid type A and glycine receptors are the major mediators of fast synaptic inhibition in the human central nervous system and are established drug targets. However, all drugs targeting these receptors bind to the extracellular ligand-binding domain of the receptors, which inherently is associated with perturbation of the basic physiological action. Here we pursue a fundamentally different approach, by instead targeting the intracellular receptor-gephyrin interaction. First, we defined the gephyrin peptide-binding consensus sequence, which facilitated the development of gephyrin super-binding peptides and later effective affinity probes for the isolation of native gephyrin. Next, we demonstrated that fluorescent super-binding peptides could be used to directly visualize inhibitory postsynaptic sites for the first time in conventional and super-resolution microscopy. Finally, we demonstrate that the gephyrin super-binding peptides act as acute intracellular modulators of fast synaptic inhibition by modulating receptor clustering, thus being conceptually novel modulators of inhibitory neurotransmission.

Super-resolution microscopyrequires small fluorescent labels.Wereport the application of genetic code expansion in combination with bioorthogonal click chemistry to label the NR1 domain of the NMDAr eceptor.W eg enerated 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.M utants were optimizedw ith regardt op rotein expression, labeling efficiency and receptor functionality as tested by fluorescence microscopyand whole-cell patchclamp. The results showt hat bioorthogonal clickc hemistry in combination with small organic dyes is superior to available immunocytochemistry protocols for receptor labeling in live and fixedc ells and enables single-molecule sensitive superresolution microscopyexperiments.

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