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

Abstract

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.

Live cell single molecule tracking and localization microscopy of bioorthogonally labeled plasma membrane proteins

“…Single Molecule Localization Microscopy (SMLM) [6][7][8][9][10][11] . However, these studies focused on the proof of concept and therefore very little quantitative information on the spatiotemporal dynamics of proteins in cells has been obtained from them.…”

A straightforward approach for bioorthogonal labeling of proteins and organelles in live mammalian cells, using a short peptide tag

“…Using genetic code expansion (GCE) and bioorthogonal chemistry, it is now possible to attach fluorescent dyes (Fl-dyes) to specific protein residues, thereby allowing direct labeling of proteins in live cells with Fl-dyes [1][2][3]. Indeed, this approach has been applied, in recent years, for fluorescent labeling of extra-and intracellular proteins [4][5][6][7][8][9][10].…”

“…In practice, however, finding a suitable labeling site can be laborious and time-consuming for several reasons. First, prior knowledge or functional assays are necessary to ensure that the insertion of the ncAA at a specific position does not affect protein structure and function [4][5][6][7]10]. Second, the efficiency of ncAA incorporation varies at different locations in the protein with no guidelines for the preferred sequence context having been reported [3][4][5][6][7]15].…”

A straightforward approach for bioorthogonal labeling of proteins and organelles in live mammalian cells, using a short peptide tag

“…Using genetic code expansion (GCE) and bioorthogonal chemistry, it is now possible to non-invasively attach fluorescent dyes (Fl-dyes) to specific protein residues, thereby allowing essentially "tag-free" labeling of proteins in live cells (1-3). Indeed, this approach has been applied, in recent years, for fluorescent labeling of extra and intra cellular proteins (4)(5)(6)(7)(8)(9)(10).…”

“…In practice, however, finding a suitable labeling site can be laborious and time-consuming for several reasons. First, prior knowledge or functional assays are necessary to ensure that insertion of the ncAA at a specific position does not affect protein structure and function (4)(5)(6)(7)10). Second, the efficiency of ncAA incorporation varies at different locations in the protein with no guidelines for the preferred sequence context having been reported (3)(4)(5)(6)(7)15).…”