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

Summary

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

Abstract

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

Introduction

The genetic incorporation of chemical probes into proteins is a powerful method to facilitate investigation of structural and dynamic aspects of protein function directly in the native context of the live cell. Nowadays, hundreds of non-canonical amino acids (ncAAs) equipped with the most disparate chemical groups can be site-specifically incorporated into proteins by biosynthesis^1,2,3,4. Between them, one finds photo-sensitive ncAAs such as photo-crosslinkers⁵, photo-caged^6,

Protocol

1. Fluorescence-based Screening of Incorporation Efficiencies (Figure 1)

1. Maintain HEK293 cells in Dulbecco's Modified Eagle's Medium (DMEM; high glucose, 4 mM glutamine, pyruvate) supplemented with 10 % (v/v) fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin at 37 °C, 95 % humidity and 5 % CO₂.
2. Seed the cells the day before transfection.
   1. Detach the cells for 5 min at 37 °C in 0.05 % Trypsin/PBS supplemented with 0.5 mM EDTA. Use 1 mL Trypsin/EDTA for a 10-cm dish. Quench with 10 volumes of complete medium and resuspend the cells by pipetting. Count the number of cells in ....

Results

The outline of the fluorescence assay is depicted in Figure 1. The assay is employed in three applications. In first place, a number of tRNA variants for incorporation of Lys(Boc) by the Pyl orthogonal pair are screened. Lys(Boc) is an amino acid sterically similar to Pyl. As Pyl is not commercially available, Lys(Boc) is commonly used as a standard substrate for the PylRS. The screened tRNAs are based on the tRNA^Pyl. Each tRNA variant bears mutati.......

Discussion

The protocol describes a simple and reliable assay to assess the efficiency of orthogonal pairs for the incorporation of ncAAs into proteins expressed in mammalian cells. The main advantage of this method in respect to widely used assays based on FACS is that it allows the simultaneous preparation and measurement of larger numbers of samples, and provides data that are easily analyzed using an ordinary software. The availability of a medium-throughput method to analyze orthogonal pairs in mammalian cells is very importan.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chemicals
Acryamide/Bisacrylamide 30% (37,5:1)	Carl Roth	3029.1
Ammonium persulfate (APS)	Carl Roth	9592.2
p-Azidophenylalanine (Azi)	Bachem	F-3075.0001
Boric acid	Sigma Aldrich	B6768
Bromphenolblue	Sigma-Aldrich	B0126-25G
Bovine serum albumine (BSA)	Carl Roth	8076.2
Carbobenzyloxy-L-lysine (Lys(Z))	NovaBiochem	8540430100
Cyclooctyne-L-lysine (SCOK)	Sichem	SC-8000
DMEM	Life Technologies	41966052
DMSO	Carl Roth	A994.2
DTT	Carl Roth	6908.1
enhanced chemiluminescence reagent (ECL)	home-made		10 mg/l luminol in 0.1 M Tris-HCl pH 8.6 ; 1100 mg/l  p-coumaric acid in DMSO ; 30 % H2O2 (1,000 : 100 : 0.3)
EDTA	Carl Roth	8043.1
EGTA	Carl Roth	3054.1
endo-bicyclo[6.1.0]nonyne-L-lysine (BCNK)	Sichem	SC-8014
FBS	Thermo Fisher (Gibco)	10270106
FluoroBrite DMEM	Thermo Fisher (Gibco)	A1896701
Glycerol	Carl Roth	7533.1
Glycin	Carl Roth	3908.3
HEPES	Carl Roth	9105.3
Hoechst 33342	Sigma Aldrich	B2261
KCl	Carl Roth	6781.3
Lipofectamine 2000	Thermo Fisher	11668019
Luminol	Applichem	A2185,0005
Methanol	Carl Roth	0082.3
MgCl2	Carl Roth	2189.2
NaCl	Carl Roth	HN00.2
Na-Lactate	Sigma-Aldrich	71718-10G
NaOH	Grüssing	121551000
PBS	Sigma-Aldrich	P5493-1L
p-Coumaric acid	Sigma-Aldrich	C9008-1G
poly-D-lysine hydrobromide	Corning	354210
PEI	Polysciences	23966
Penicillin/Streptomycin	Thermo Fisher (Gibco)	11548876 (15140-122)
PMSF	Carl Roth	6367.1
PNGase F	NEB	P0704L
Protease Inhibitor	Roche	11873580001
PVDF membrane Immobilon-P	Millipore	IPVH00010
Skim Milk Powder	Sigma	70166
Sodium dodecyl sulfate (SDS)	Carl Roth	CN30.2
Tetrazine-Cy3	Jena Bioscience	CLK-014-05
Tetramethylethylenediamine (TEMED)	Carl Roth	2367.3
trans-Cyclooctene-L-lysine (TCO*K)	Sichem	SC-8008
TRIS	Sigma-Aldrich	T1503
Triton X-100	Carl Roth	3051.4
Trypsin 2.5%	Thermo Fisher (Gibco)	15090046
Tween 20	Carl Roth	9127.2
Wasserstoffperoxid (30%)	Merck	1.07210.0250
Cell lines
HEK293 cells	German Collection of Microorganisms and Cell Cultures GmbH (DSMZ)	ACC-305
HEK293T cells	German Collection of Microorganisms and Cell Cultures GmbH (DSMZ)	ACC-635
Equipment
Crosslinker Bio-Link 365 nm	Bio-Budget Technologies GmbH	40-BLX-E365	5 x 8 Watt tubes
Plate Reader BMG LABTECH FLUOstar Omega	BMG LABTECH
Plasmids
Plasmid E2AziRS	The huminized gene for E2AziRS was synthesized by Geneart (Life Technologies)		Plasmid containing 4 tandem copies of the suppressor tRNA Bst-Yam driven by the human U6 promoter and one copy of a humanized gene for the enhanced variant of the Azi-tRNA synthetase (EAziRS) driven by a PGK promoter
POI-TAG mutant plasmids			Plasmid encoding the POI driven by the CMV promoter, C-terminally fused to the FLAG-tag, bearing a TAG codon at the desired position
CRF1R-95TAG-EGFP			Cloned in the MCS of pcDNA3.1
HA-PTH1R-79TAG-CFP			Cloned in the MCS of pcDNA3.1
Arrestin3-FLAG	Synthesized by Genart (Life Technologies)		Cloned in the MCS of pcDNA3.1
Antibodies
Anti-FLAG-HRP M2 antibody conjugate	Sigma-Aldrich	A8592	monoclonal, produced in mouse clone M2
Goat-anti-rabbit-HRP antibody	Santa Cruz	sc-2004
Rabbit-anti-CRF antibody	home-made	PBL #rC69	polyclonal Turnbull, A.V., Vaughan, J., Rivier, J.E., and Vale, W.W. Endocrinology, 140, (1), 71-78 (1999)
Rabbit-anti-Ucn1 antibody	home-made	PBL #5779	polyclonal Turnbull, A.V., Vaughan, J., Rivier, J.E., and Vale, W.W. Endocrinology, 140, (1), 71-78 (1999)