Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

Serfling, Robert; Seidel, Lisa; Böttke, Thore; Coin, Irene

doi:10.3791/57069

Cited by 5 publications

(7 citation statements)

References 71 publications

(97 reference statements)

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“…Cells were cotransfected with MbPylRS AF /4xtRNA M15 and receptor constructs in a 1:1 ratio for a total amount of 1.5 μg of DNA using Lipofectamine 2000 (Thermo Fisher Scientific) as described. 48 Labeling Procedure. The glass covers were transferred 16−20 h post-transfection to new 6-well plates containing complete dye-free FluoroBrite medium (Thermo Fisher Scientific), and cells were incubated at least 2 h without ncAA at 37 °C under CO 2 (5%) atmosphere.…”

Section: ■ Methodsmentioning

confidence: 99%

“…TCO* (SiChem, Bremen, Germany) was added 1 h before transfection to a concentration of 250 μM for extracellular labeling and 50 μM for intracellular labeling experiments. Cells were cotransfected with Mb PylRS AF /4xtRNA M15 and receptor constructs in a 1:1 ratio for a total amount of 1.5 μg of DNA using Lipofectamine 2000 (Thermo Fisher Scientific) as described …”

Section: Methodsmentioning

confidence: 99%

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Quantitative Single-Residue Bioorthogonal Labeling of G Protein-Coupled Receptors in Live Cells

et al. 2019

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High-end microscopy studies of G protein-coupled receptors (GPCRs) require installing onto the receptors bright and photostable dyes. Labeling must occur in quantitative yields, to allow stoichiometric data analysis, and in a minimally invasive fashion, to avoid perturbing GPCR function. We demonstrate here that the genetic incorporation of trans-cyclooct-2-ene lysine (TCO*) allows achieving quantitative single-residue labeling of the extracellular loops of the β2-adrenergic and the muscarinic M2 class A GPCRs, as well as of the corticotropin releasing factor class B GPCR. Labeling occurs within a few minutes by reaction with dye–tetrazine conjugates on the surface of live cells and preserves the functionality of the receptors. To precisely quantify the labeling yields, we devise a method based on fluorescence fluctuation microscopy that extracts the number of labeling sites at the single-cell level. Further, we show that single-residue labeling is better suited for studies of GPCR diffusion than fluorescent-protein tags, since the latter can affect the mobility of the receptor. Finally, by performing dual-color competitive labeling on a single TCO* site, we devise a method to estimate the oligomerization state of a GPCR without the need for a biological monomeric reference, which facilitates the application of fluorescence methods to oligomerization studies. As TCO* and the dye–tetrazines used in this study are commercially available and the described microscopy techniques can be performed on a commercial microscope, we expect our approach to be widely applicable to fluorescence microscopy studies of membrane proteins in general.

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Section: ■ Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Quantitative Single-Residue Bioorthogonal Labeling of G Protein-Coupled Receptors in Live Cells

et al. 2019

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“…The secondary antibody, goat anti‐rabbit IgG‐HRP (SantaCruz #sc‐2004, 1:15 000) or the mouse‐anti‐FLAG M2‐HRP conjugate (Sigma, 1:5000), was applied for 1 h at RT, followed by 3×10 min washes in TBS‐T. Homemade ECL reagent (0.1 m Tris ⋅ HCl pH 8.6, 1 m m luminol, 0.6 m m p ‐coumaric acid, 10 % DMSO, 0.01 % H 2 O 2 ) was applied to the membranes for 1 min and signals were collected for 5 min in the dark (Gbox, Syngene). All western blots were replicated at least twice in independent experiments.…”

Section: Methodsmentioning

confidence: 99%

Exploring Pairwise Chemical Crosslinking To Study Peptide–Receptor Interactions

et al. 2019

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Pairwise crosslinking is a powerful technique to characterize interactions between G protein coupled receptors and their ligands in the live cell. In this work, the “thiol trapping” method, which exploits the proximity‐enhanced reaction between haloacetamides and cysteine, is examined to identify intermolecular pairs of vicinal positions. By incorporating cysteine into the corticotropin‐releasing factor receptor and either α‐chloro‐ or α‐bromoacetamide groups into its ligands, it is shown that thiol trapping provides highly reproducible signals and a low background, and represents a valid alternative to classical “disulfide trapping”. The method is advantageous if reducing agents are required during sample analysis. Moreover, it can provide partially distinct spatial constraints, thus giving access to a wider dataset for molecular modeling. Finally, by applying recombinant mini‐Gs, GTPγS, and Gαs‐depleted HEK293 cells to modulate Gs coupling, it is shown that yields of crosslinking increase in the presence of elevated levels of Gs.

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“…Installing artificial probes and reactive moieties directly onto proteins in the live cell is also an interesting objective that could be achieved through the incorporation of NSAAs. A robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells enabled the screening of tens to hundreds of orthogonal aaRS/tRNA variants (Serfling et al 2018). This approach was used both to incorporate chemical probes into G-proteincoupled receptors for photo-crosslink mapping and to achieve biocatalyst-free receptor labeling with a fluorescent dye via biorthogonal chemistry in the live cell.…”

Section: Real-time Tracking and Imaging In Vivomentioning

confidence: 99%

Incorporation of non-standard amino acids into proteins: challenges, recent achievements, and emerging applications

Jin

Park

Hong

2019

Appl Microbiol Biotechnol

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The natural genetic code only allows for 20 standard amino acids in protein translation, but genetic code reprogramming enables the incorporation of non-standard amino acids (NSAAs). Proteins containing NSAAs provide enhanced or novel properties and open diverse applications. With increased attention to the recent advancements in synthetic biology, various improved and novel methods have been developed to incorporate single and multiple distinct NSAAs into proteins. However, various challenges remain in regard to NSAA incorporation, such as low yield and misincorporation. In this review, we summarize the recent efforts to improve NSAA incorporation by utilizing orthogonal translational system optimization, cell-free protein synthesis, genomically recoded organisms, artificial codon boxes, quadruplet codons, and orthogonal ribosomes, before closing with a discussion of the emerging applications of NSAA incorporation.

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Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

Cited by 5 publications

References 71 publications

Quantitative Single-Residue Bioorthogonal Labeling of G Protein-Coupled Receptors in Live Cells

Quantitative Single-Residue Bioorthogonal Labeling of G Protein-Coupled Receptors in Live Cells

Exploring Pairwise Chemical Crosslinking To Study Peptide–Receptor Interactions

Incorporation of non-standard amino acids into proteins: challenges, recent achievements, and emerging applications

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