Name: optimizing the genetic incorporation of chemical probes into gpcrs for photo-crosslinking mapping and bioorthogonal chemistry in live mammalian cells
Uploaded: 2018-04-09T14:30:00
Description: Watch this Scientific Journal Video about Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells at JoVE.

This work has been founded by the Deutsche Forschungsgemeinschaft (DFG) under grants CO822/2-1 (Emmy-Noether program) and CO822/3-1 to I.C.

1. Fluorescence-based Screening of Incorporation Efficiencies (Figure 1)
<ol>
	<li>Maintain HEK293 cells in Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM; high glucose, 4 mM glutamine, pyruvate) supplemented with 10 % (v/v) fetal bovine serum (FBS), 100 U/mL penicillin and 100 &#181;g/mL streptomycin at 37 &#176;C, 95 % humidity and 5 % CO2.</li>
	<li>Seed the cells the day before transfection.
	<ol>
		<li>Detach the cells for 5 min at 37 &#176;C in 0.05 % Trypsin/PBS supplem...

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...

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 biosynthesis1,2,3,4. Between them, one finds photo-sensitive ncAAs such as photo-crosslinkers5, photo-caged6,7,8,9 and photo-switchable amino acids10,11, amino acids bearing strained alkenes and alkynes for catalyst-free bioorthogonal chemistry2,12,13,14,15,16,17, amino acids carrying dansyl18, coumarin9,19, and prodan20,21 fluorophores, and amino acids equipped with other biophysical probes as well as with post translational modifications1,2,3,4,22,23,24,25.
The genetic encoding of a ncAA is enabled by a dedicated amino-acyl-tRNA-synthetase (AARS) paired to a cognate suppressor-tRNA, which incorporates the ncAA in response to an amber stop codon during the regular ribosomal synthesis. ncAARS/tRNA pairs are engineered so as to be orthogonal in the host organism, i.e. not cross-talk with the endogenous pairs. The technique is well established both in prokaryotic and eukaryotic hosts and easily applicable to mammalian cells. Pairs for ncAA incorporation in mammalian cells are based on three main orthogonal systems: the tyrosyl system, that combines the TyrRS from E. coli26 with a tyrosyl amber suppressor from B. stearothermophilus27 (EcTyrRS/BstYam pair), the E. coli leucyl system (EcLeuRS/tRNALeuCUA pair)6,18,28 and the archaeal pyrrolysyl system (PylRS/tRNAPyl pair)3, whereby the tRNAPyl is a natural amber suppressor. In general, each ncAA is recognized by a specialized ncAARS. Depending on the structure of the ncAA, the ncAARS is obtained via directed evolution of either TyrRS, LeuRS or PylRS, although some synthetases can accept more than one ncAA.
The orthogonal pair is imported into the cells by simply using a plasmid vector. Most common and efficient plasmids are bicistronic and encode both for the synthetase and the tRNA forming the orthogonal pair29. A second plasmid encoding for the protein of interest bearing an amber codon at the site designated for modification is co-transfected. The ncAA is simply added to the cell growth medium. However, different specialized groups often use different variants of plasmid constructs even for the incorporation of the same ncAA. Constructs differ in the arrangement of the genes in the vector, type of the synthetase, codon usage in the synthetase gene, promoter usage, variant of the tRNA and number of tRNA expression cassettes. Moreover, the incorporation efficiency of different ncAAs can vary drastically due to the different catalytic efficiency of the different synthetases, the quality of the tRNA, and other factors30. Therefore, it is important to have at hand a fast and reliable method to evaluate the efficiency of an orthogonal pair, both to choose the most suitable system for a desired application and to perform some optimization steps that improve overall protein expression yields.
We have established a simple and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs29 (Figure 1). In the assay, cells are co-transfected with the plasmid encoding for the orthogonal pair, together with a bicistronic reporter plasmid encoding both for the green fluorescent protein bearing an amber stop codon at a permissive position (EGFPTAG) and the mCherry gene. Red and green fluorescence of whole-cell lysates are read in separate channels on a plate reader in a 96-well plate. The intensity of the green fluorescence directly correlates with the efficiency of amber suppression, whereas the intensity of red fluorescence gives a direct estimate of the size of the measured sample and the transfection efficiency. With respect to similar assays based on fluorescence assisted cell sorting (FACS) read out31,32, the assay gives an immediate and comprehensive assessment of protein expression in the whole cell population, which is more representative of usual experimental conditions, and offers an easier data acquisition and processing with standard software. Overall, the main advantage of the assay is that a medium to a large number of samples can be analyzed in parallel. Using this assay, we have screened a rationally designed library of suppressor-tRNAs to improve the efficiency of the Pyl orthogonal system30. This work describes the experimental protocol to perform this assay and show examples of its application, including the optimization of the orthogonal pair for the incorporation of the photo-crosslinking ncAA p-azido-L-phenylalanine (Azi) and the comparison of incorporation efficiencies of different amino acids (Figure 2).
Over the last years, ncAA tools have been proven very powerful to investigate structural and functional aspects of G-protein coupled receptors (GPCRs)33,34,35,36,37,38. In humans, GPCRs form a large family of membrane receptors (800 members) and represent main targets for therapeutic drugs. Direct structural characterization of GPCRs is still challenging and complementary biochemical methods are highly needed for their investigation. We have pioneered the use of photo-crosslinking ncAAs to map GPCR surfaces and discover ligand binding pockets34. Using our optimized system for Azi incorporation, we systematically incorporated Azi throughout the whole juxtamembrane domain of a GPCR directly in live mammalian cells. Upon UV irradiation, Azi forms a highly reactive nitrene species that covalently captures neighboring molecules. When the ligand is added to the system, Azi serves as a proximity probe to reveal which positions of the receptor come close to the bound ligand. In this way, the binding mode of the neuropeptide hormone Urocortin I (Ucn1) on the class B GPCR corticotropin-releasing-factor receptor type 1 (CRF1R)33 was first unveiled. Lately, we have disclosed distinct binding patterns of agonists and antagonists on the same receptor38. A similar approach has been applied by others to reveal orthosteric and allosteric binding sites of other peptides and small molecule ligands on other GPCRs39,40,41,42. This manuscript describes the experimental protocol applied in our lab for photo-crosslinking mapping of GPCR surfaces. The method is relatively fast, straightforward and does not require any special equipment, so that it is applicable in standard biochemistry labs. Importantly, the approach provides a valuable tool not only to identify ligand binding sites where 3D structural data are scarce, but also to supplement existing in vitro data with information from fully post-translationally modified receptors in the physiological environment of the live cell.
The recent development of novel ncAAs bearing on the side chain chemical groups suitable for ultrafast catalyst-free bioorthogonal chemistry has opened up the possibility to install last-generation fluorophores for super-resolution imaging into proteins directly on the live cells2,43. Such chemical anchors include strained cyclooctyne in SCOK14, bicyclo[6.1.0]nonyne in BCNK12,17, and trans-cyclooctenes in TCO*K13,15,17 among other ncAAs harboring a norbornene16,17,44 or cyclopropene45,46 moiety. Bulky ncAAs for bioorthogonal chemistry are incorporated by a variant of the PylRS usually denoted as PylRSAF (indicating mutation Y271A and Y349F in M. barkeri PylRS), as well as by other ad hoc evolved ncAARSs17,44. The bioorthogonal anchors react with tetrazine reagents47 via inverse electron-demand Diels-Alder cycloaddition to give high labeling yields within a few minutes43,48. However, application of this powerful approach to label GPCRs has been challenging due to a low overall efficiency of the orthogonal ncAA incorporation system. Using our enhanced Pyl system, we have recently demonstrated high-yield incorporation of such amino acids into GPCRs and ultrafast GPCR labeling on the surface of live mammalian cells30. Labeled receptors were still functional, as they physiologically internalized upon activating the receptor with an agonist. The experimental protocol for the incorporation of bioorthogonal anchors into GPCRs and the following labeling steps are described here. Equipping GPCRs with small bright fluorophores is the first fundamental step toward the study of GPCR structural dynamics in the live cell via advanced microscopy techniques.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acryamide/Bisacrylamide 30% (37,5:1)</td>
			<td>Carl Roth</td>
			<td>3029.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>Carl Roth</td>
			<td>9592.2</td>
			<td></td>
		</tr>
		<tr>
			<td>p-Azidophenylalanine (Azi)</td>
			<td>Bachem</td>
			<td>F-3075.0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Sigma Aldrich</td>
			<td>B6768</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromphenolblue</td>
			<td>Sigma-Aldrich</td>
			<td>B0126-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumine (BSA)</td>
			<td>Carl Roth</td>
			<td>8076.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbobenzyloxy-L-lysine (Lys(Z))</td>
			<td>NovaBiochem</td>
			<td>8540430100</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclooctyne-L-lysine (SCOK)</td>
			<td>Sichem</td>
			<td>SC-8000</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>41966052</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Carl Roth</td>
			<td>A994.2</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Carl Roth</td>
			<td>6908.1</td>
			<td></td>
		</tr>
		<tr>
			<td>enhanced chemiluminescence reagent (ECL)&#160;</td>
			<td>home-made</td>
			<td></td>
			<td>10 mg/l luminol in 0.1 M Tris-HCl pH 8.6 ; 1100 mg/l&#160; p-coumaric acid in DMSO ; 30&#160;% H2O2 (1,000 : 100 : 0.3) [Quelle Laborjounal]</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Carl Roth</td>
			<td>8043.1</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Carl Roth</td>
			<td>3054.1</td>
			<td></td>
		</tr>
		<tr>
			<td>endo-bicyclo[6.1.0]nonyne-L-lysine (BCNK)</td>
			<td>Sichem</td>
			<td>SC-8014</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Thermo Fisher (Gibco)</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>FluoroBrite DMEM</td>
			<td>Thermo Fisher (Gibco)</td>
			<td>A1896701</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Carl Roth</td>
			<td>7533.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycin</td>
			<td>Carl Roth</td>
			<td>3908.3</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Carl Roth</td>
			<td>9105.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Sigma Aldrich</td>
			<td>B2261</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Carl Roth</td>
			<td>6781.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000&#160;</td>
			<td>Thermo Fisher</td>
			<td>11668019</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminol</td>
			<td>Applichem</td>
			<td>A2185,0005</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Carl Roth</td>
			<td>0082.3</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Carl Roth</td>
			<td>2189.2</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Carl Roth</td>
			<td>HN00.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Na-Lactate</td>
			<td>Sigma-Aldrich</td>
			<td>71718-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Gr&#252;ssing</td>
			<td>121551000</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>P5493-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>p-Coumaric acid</td>
			<td>Sigma-Aldrich</td>
			<td>C9008-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>poly-D-lysine hydrobromide</td>
			<td>Corning</td>
			<td>354210</td>
			<td></td>
		</tr>
		<tr>
			<td>PEI</td>
			<td>Polysciences</td>
			<td>23966</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo Fisher (Gibco)</td>
			<td>11548876 (15140-122)</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Carl Roth</td>
			<td>6367.1</td>
			<td></td>
		</tr>
		<tr>
			<td>PNGase F</td>
			<td>NEB</td>
			<td>P0704L</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF membrane Immobilon-P</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Skim Milk Powder&#160;</td>
			<td>Sigma</td>
			<td>70166</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Carl Roth</td>
			<td>CN30.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrazine-Cy3</td>
			<td>Jena Bioscience</td>
			<td>CLK-014-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylethylenediamine (TEMED)</td>
			<td>Carl Roth</td>
			<td>2367.3</td>
			<td></td>
		</tr>
		<tr>
			<td>trans-Cyclooctene-L-lysine (TCO*K)</td>
			<td>Sichem</td>
			<td>SC-8008</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIS</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Carl Roth</td>
			<td>3051.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 2.5%</td>
			<td>Thermo Fisher (Gibco)</td>
			<td>15090046</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Carl Roth</td>
			<td>9127.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Wasserstoffperoxid (30%)</td>
			<td>Merck</td>
			<td>1.07210.0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell lines</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293 cells</td>
			<td>German Collection of Microorganisms and Cell Cultures GmbH (DSMZ)</td>
			<td>ACC-305</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T cells</td>
			<td>German Collection of Microorganisms and Cell Cultures GmbH (DSMZ)</td>
			<td>ACC-635</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Crosslinker Bio-Link 365 nm</td>
			<td>Bio-Budget Technologies GmbH</td>
			<td>40-BLX-E365</td>
			<td>5 x 8 Watt tubes</td>
		</tr>
		<tr>
			<td>Plate Reader BMG LABTECH FLUOstar Omega&#160;</td>
			<td>BMG LABTECH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid E2AziRS</td>
			<td>The huminized gene for E2AziRS was synthesized by Geneart (Life Technologies)</td>
			<td></td>
			<td>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</td>
		</tr>
		<tr>
			<td>POI-TAG mutant plasmids</td>
			<td></td>
			<td></td>
			<td>Plasmid encoding the POI driven by the CMV promoter, C-terminally fused to the FLAG-tag, bearing a TAG codon at the desired position&#160;</td>
		</tr>
		<tr>
			<td>CRF1R-95TAG-EGFP</td>
			<td></td>
			<td></td>
			<td>Cloned in the MCS of pcDNA3.1</td>
		</tr>
		<tr>
			<td>HA-PTH1R-79TAG-CFP</td>
			<td></td>
			<td></td>
			<td>Cloned in the MCS of pcDNA3.1</td>
		</tr>
		<tr>
			<td>Arrestin3-FLAG</td>
			<td>Synthesized by Genart (Life Technologies)</td>
			<td></td>
			<td>Cloned in the MCS of pcDNA3.1</td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-FLAG-HRP M2 antibody conjugate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A8592&#160;</td>
			<td>monoclonal, produced in mouse clone M2</td>
		</tr>
		<tr>
			<td>Goat-anti-rabbit-HRP antibody</td>
			<td>Santa Cruz</td>
			<td>sc-2004</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit-anti-CRF antibody</td>
			<td>home-made</td>
			<td>PBL #rC69</td>
			<td>polyclonal [Turnbull]</td>
		</tr>
		<tr>
			<td>Rabbit-anti-Ucn1 antibody</td>
			<td>home-made</td>
			<td>PBL #5779</td>
			<td>polyclonal [Turnbull]</td>
		</tr>
	</tbody>
</table>

optimizing the genetic incorporation of chemical probes into gpcrs for photo-crosslinking mapping and bioorthogonal chemistry in live mammalian cells

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.

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 tRNAPyl. Each tRNA variant bears mutati...

Watch this Scientific Journal Video about Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells at JoVE.

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

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.

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

The overall goal of this protocol is to enhance the incorporation rate of non-canonical amino acids in mammalian cells to tackle biological questions in live cell settings including mapping of protein-protein interaction surfaces, and bioorthogonal labeling of surface receptors. Using amber codon suppression, different non-canonical amino acids are incorporated with different efficiencies. We established fluorescence assay to easily compare non-canonical amino acids incorporation rates in mammalian cells within a short time.The systematic incorporation of photo-crosslinking, non-canonical amino acids through our GPCR surfaces allows mapping of ligand binding sites and provides precious information of the intake receptor directly from the life cell. By incorporating last generation, non-canonical amino acids for bioorthogonal chemistry, we achieve site-specific, super fast, catalyst free labeling of GPCRs with small, bright fluorophores for high-end imaging applications in live mammalian cells. First, seed 600, 000 HEK 293 cells in two milliliters of complete growth medium per well in two six-well plates.Prepare as many wells as the number of samples and two additional wells for the wild type EGFP and a mock transfected sample respectively. One hour prior to transfection, add the appropriate amount of freshly prepared non-canonical amino acid stock solution to all wells for a final amino acid concentration of 0.25 to 0.5 millimolar. To transfect the cells, mix one microgram of plasmid DNA and coding for the synthetase t-RNA pair to be tested with one microgram of reporter plasmid DNA in a microcentrifuge tube.In separate tubes, prepare an identical transfection using the EGFP wild type reference and a mock transfection. Next, add 100 microliters of lactate buffered saline to each tube containing the DNA. After briefly mixing, add six microliters of one microgram per microliter PEI and lactate buffered saline to each tube.Vortex immediately, and then incubate at room temperature for 10 to 15 minutes. Transfer 400 microliters of cell medium from each well to the DNA PEI mixture to neutralize the pH. Then, dribble the DNA mixture onto the cells.Harvest the cells 48 hours post-transfection by aspirating the medium and rinsing the cells once with two milliliters of pre-warmed PBS. Add 800 microliters of PBS supplemented with 0.5 millimolar EDTA, and incubate for 20 minutes at 37 degrees Celsius. After incubation, detach and suspend the cells by pipetting up and down.Next, transfer the cell suspension into microcentrifuge tubes containing 200 microliters of PBS supplemented with five millimolar magnesium chloride. Centrifuge the samples for two minutes at 800 times gravity. When finished, discard the supernatant.Now, add 100 microliters of Tris lysis buffer to the cell pellets and incubate on ice for 30 minutes. To facilitate lysis, vortex every five minutes. Spin down the cell debris for 10 minutes at four degrees Celsius and 14, 000 times gravity.Transfer 90 microliters of the supernatant into black 96-well plates. Then, measure EGFP and mCherry fluorescence using a plate reader equipped with the fluorescence module. Seed 500, 000 293 t-cells in two milliliters of complete growth medium per well in two six-well plates.For each position to be screened, prepare one well per ligand, plus one well for the binding control. One hour prior to transfection, add the photo-crosslinker, p-azidophenylalanine, or Azi, to all wells to a final concentration of 0.5 millimolar. Transfect a total amount of two micrograms of DNA per well using one microgram of plasmid and coding for the flag tagged GPCR bearing a tag codon at the desired position, and one microgram of the plasmid and coding for the orthogonal pair dedicated to Azi.At 48 hours post-transfection, prepare a 1, 000x ligand stock solution by dissolving the peptide ligand at a concentration of 100 micromolar in DMSO. Dilute the ligand stock solution one to 1, 000 in binding buffer. Replace the cell medium with one milliliter of the ligand solution and incubate the samples for 10 minutes at room temperature.Following incubation, irradiate the cells for 20 minutes in a UV cross-linker at 365 nanometers and five centimeter distance to the cells. When finished, detach the cells by pipetting and transfer them into a microcentrifuge tube. Pellet the cells for three minutes at 800 times gravity.After discarding the supernatant, resuspend the cell pellets in 50 microliters of HEPES dissociation buffer, or HDB, containing a cocktail of protease inhibitors. Then, flash freeze the cells in liquid nitrogen. Next, thaw the cells in a water bath at 37 degrees Celsius for 30 to 45 seconds, and then vortex briefly.Pellet the membranes for 10 minutes at four degrees Celsius and 2500 times gravity. When finished, discard the supernatant which contains the bulk of cytosolic proteins. Thoroughly resuspend the pellets in 50 microliters of HEPES lysis buffer using a pipet.Then, lyse the cells for 30 minutes on ice, vortexing every five minutes. Following this, spin down the cell debris for 10 minutes at four degrees Celsius and 14, 000 times gravity. Immediately transfer each supernatant to a fresh reaction tube for western blot analysis.When the GPCR is glycosylated and faint or smeared bands are a problem in western blot analysis, deglycosylate the samples with PNGase F to increase signal intensity and sharpen the bands. Then, resolve the samples on standard SDS page and blot transfer proteins to a PVDF membrane. After blocking the membrane, probe it with an anti-ligand antibody to detect the occurrence of cross-linking.To detect the expression level of the Azi-GPCR, probe the membrane with a commercial anti-flag antibody. After washing the membrane, soak it in enhanced chemiluminescence solution, and visualize the signals in the dark. Seed 100, 000 HEK 293 t-cells in 600 microliters of dye-free, complete DMEM per well of a microscopy slide equipped with four wells.One hour prior to transfection, prepare a fresh 100 millimolar stock solution of TCOK and 0.2 molar sodium hydroxide and 15%DMSO. Mix three microliters of the TCOK stock solution with 12 microliters of one molar, pH 7.4 HEPES buffer. Gently add the solution to each well for a final TCOK concentration of 0.5 millimolar.In a microcentrifuge tube, dilute 200 nanograms of the plasmid and coding for the receptor containing the amber codon and 200 nanograms of the plasmid and coding for the orthogonal pair in 50 microliters of medium. Dilute 1.25 microliters of lipofectamine 2, 000 in 50 microliters of dye-free, serum-free, and antibiotic-free medium, and add the solution to the DNA solution. Vortex immediately and incubate for five to 10 minutes at room temperature.Then, add the DNA lipid complexes to the cells. At 24 hours post-transfection, transfer 100 microliters of medium from each well into a 1.5 milliliter reaction tube. Add 1.8 microliters of an orange fluorescent dye tetrazine stock solution, and 0.3 microliters of a DNA staining dye stock solution.Transfer the medium containing the dyes back to the well and incubate for five minutes at 37 degrees Celsius. Aspirate the medium, and gently rinse the cells twice with PBS to remove the excess dye. Then, add 600 microliters of pre-heated, complete dye-free growth medium to the cells.Now, visualize the labeled receptors under 63x magnification using filters appropriate for GFP orange fluorescent dye and DNA-staining dye. After adding peptide agonist stock solution to the cells, observe the internalization under the microscope using the aforementioned filters and take pictures after the clearly detectable occurrence of internalization. Different tRNA's give different suppression efficiency, which is clearly reflected in the amount of measured fluorescence.Azi incorporation into CRF1R was highly enhanced when using the codon-optimized Azi-tRNA synthetase gene compared to the native gene, showing that even a moderate improvement for a soluble protein can have a greater impact on the expression level of more challenging membrane proteins. The optimized system for Azi incorporation, including the humanized E2 Azi RS gene was deployed to map the binding pocket of the CRF1R and to find binding paths of five ligands. A band at the correct size of the crosslinking product in the western blot reveals that the position lies in the proximity of the ligand within the ligand receptor complex.Multiple crosslinking hits with all ligands tested revealed distinct binding patterns for the peptide Agonists and Antagonists. Both fluorescence and western blot data suggests that TCOK is the non-canonical amino acids for bioorthogonal chemistry that gets incorporated with the highest efficiency of the AF variant of the paralysine tRNA synthetase. TCOK was incorporated into a CRF1R EGFP fusion protein and enabled installing a small, bright fluorophore on the receptor.After adding a peptide Agaonist, fluorescent compartments were observed throughout the cytosol, revealing the physiological process of GPCR internalization. To do a fluorescence assay, we presented here is a simple and effective tool to evaluate and optimize the efficiency of a synthetase TI and API in mammalian cells. High incorporation rates are crucial for applying non-canonical amino acids to investigate challenging biological questions.Highly efficient incorporation of crosslinking non-canonical amino acids enables extensive surface mapping of virtually any protein, including membrane receptors or low-banded proteins. The method reveals the topology of interaction interfaces which can be further analyzed to obtain accurate molecular models. High incorporation efficiency of last generation, non-canonical amino acids for bioorthogonal chemistry allows us to equip GPCRs with small, bright fluorophores, thus leading the way towards the study of GPCRs structural dynamics in live cells via advanced microscopy techniques.

optimizing-genetic-incorporation-chemical-probes-into-gpcrs-for-photo

Research

JoVE Journal

Chemistry

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.

Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal fermentation processes and screening for new oil-rich strains requires a fast and reliable protocol for determination of intracellular lipid content. Current practices rely largely on gravimetric methods to determine oil content, techniques developed decades ago that are time consuming and require large sample volumes. In this paper, Nile Red, a fluorescent dye that has been used to identify the presence of lipid bodies in numerous types of organisms, is incorporated into a simple, fast, and reliable protocol for measuring the neutral lipid content of Auxenochlorella protothecoides, a green alga. The method uses ethanol, a relatively mild solvent, to permeabilize the cell membrane before staining and a 96 well micro-plate to increase sample capacity during fluorescence intensity measurements. It has been designed with the specific application of monitoring bioprocess performance. Previously dried samples or live samples from a growing culture can be used in the assay.

A simple protocol to determine the neutral lipid content of algal cells using a Nile Red staining procedure is described. This time-saving technique offers an alternative to traditional gravimetric-based lipid quantification protocols. It has been designed for the specific application of monitoring bioprocess performance.

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal ...

Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over large or small sample areas. It has been applied in many areas of science, including materials science, geoscience, studying works of cultural heritage, and in chemical biology. In the case of chemical biology, for example, visualizing the metal distributions within cells allows us to study both naturally-occurring metal ions in the cells, as well as exogenously-introduced metals such as drugs and nanoparticles. Due to the fully hydrated nature of nearly all biological samples, cryo-fixation followed by imaging under cryogenic temperature represents the ideal imaging modality currently available. However, under the circumstances that such a combination is not easily accessible or practical, aldehyde based chemical fixation remains useful and sometimes inevitable. This article describes in as much detail as possible in the preparation of adherent mammalian cells by chemical fixation for X-ray fluorescent imaging.

Here, we present a protocol on how to determine the quantity and distribution of metals in a sample using synchrotron X-ray fluorescence. We focus on adherent cells, and describe the chemical fixation method to prepare this sample. We then describe how to mount and image the sample using synchrotron X-rays.

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over ...

Chemical Vapor Deposition of an Organic Magnet, Vanadium Tetracyanoethylene

Recent progress in the field of organic materials has yielded devices such as organic light emitting diodes (OLEDs) which have advantages not found in traditional materials, including low cost and mechanical flexibility. In a similar vein, it would be advantageous to expand the use of organics into high frequency electronics and spin-based electronics. This work presents a synthetic process for the growth of thin films of the room temperature organic ferrimagnet, vanadium tetracyanoethylene (V[TCNE]x, x~2) by low temperature chemical vapor deposition (CVD). The thin film is grown at &#60;60 &#176;C, and can accommodate a wide variety of substrates including, but not limited to, silicon, glass, Teflon and flexible substrates. The conformal deposition is conducive to pre-patterned and three-dimensional structures as well. Additionally this technique can yield films with thicknesses ranging from 30 nm to several microns. Recent progress in optimization of film growth creates a film whose qualities, such as higher Curie temperature (600 K), improved magnetic homogeneity, and narrow ferromagnetic resonance line-width (1.5 G) show promise for a variety of applications in spintronics and microwave electronics.

We present the synthesis of the organic-based ferrimagnet vanadium tetracyanoethylene (V[TCNE]x, x~2) via low temperature chemical vapor deposition (CVD). This optimized recipe yields an increase in Curie temperature from 400 K to over 600 K and a dramatic improvement in magnetic resonance properties.

Recent progress in the field of organic materials has yielded devices such as organic light emitting diodes (OLEDs) which have advantages not found in ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst immobilization for improved production yield and sustained synthesis. Described herewith is the experimental procedure for the development of such a system consisting of two E. coli strains that express functionally complementary enzymes. Together, these two enzymes can function co-operatively to mediate the regeneration of expensive cofactors for improving the product yield of the bioreaction. In addition, the method of synthesizing an immobilized form of the coupled biocatalytic system by encapsulation of whole cells in calcium alginate beads is reported. As an example, we present the improved biosynthesis of L-xylulose from L-arabinitol by coupling E. coli cells expressing the enzymes L-arabinitol dehydrogenase or NADH oxidase. Under optimal conditions and using an initial concentration of 150 mM L-arabinitol, the maximal L-xylulose yield reached 96%, which is higher than those reported in the literature. The immobilized form of the coupled whole-cell biocatalysts demonstrated good operational stability, maintaining 65% of the yield obtained in the first cycle after 7 cycles of successive re-use, while the free cell system almost completely lost the catalytic activity. Therefore, the methods reported here provides two strategies that could help improve the industrial production of L-xylulose, as well as other value-added compounds requiring the use of cofactors in general.

Presented is the protocol for co-immobilizing whole-cell biocatalysts for cofactor regeneration and improved reusability, using the production of L-xylulose as an example. The cofactor regeneration is achieved by coupling two Escherichia coli strains expressing functionally complementary enzymes; the whole-cell biocatalyst immobilization is achieved by cell encapsulation in calcium alginate beads.

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

The Preparation and Properties of Thermo-reversibly Cross-linked Rubber Via Diels-Alder Chemistry

A method for using Diels Alder thermo-reversible chemistry as cross-linking tool for rubber products is demonstrated. In this work, a commercial ethylene-propylene rubber, grafted with maleic anhydride, is thermo-reversibly cross-linked in two steps. The pending anhydride moieties are first modified with furfurylamine to graft furan groups to the rubber backbone. These pendant furan groups are then cross-linked with a bis-maleimide via a Diels-Alder coupling reaction. Both reactions can be performed under a broad range of experimental conditions and can easily be applied on a large scale. The material properties of the resulting Diels-Alder cross-linked rubbers are similar to a peroxide-cured ethylene/propylene/diene rubber (EPDM) reference. The cross-links break at elevated temperatures (&#62; 150 &#176;C) via the retro-Diels-Alder reaction and can be reformed by thermal annealing at lower temperatures (50-70 &#176;C). Reversibility of the system was proven with infrared spectroscopy, solubility tests and mechanical properties. Recyclability of the material was also shown in a practical way, i.e., by cutting a cross-linked sample into small parts and compression molding them into new samples displaying comparable mechanical properties, which is not possible for conventionally cross-linked rubbers.

A simple two-step approach involving rubber modification and cross-linking yields fully reworkable, elastic rubber products.

A method for using Diels Alder thermo-reversible chemistry as cross-linking tool for rubber products is demonstrated. In this work, a commercial ...

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals leads to the formation of composite materials with unique properties. In addition, the undesirable process of biofouling is initiated by the adsorption of biomolecules, mainly proteins, on surfaces. This organic layer is an adhesion layer for bacteria and allows them to interact with the surface. Understanding the fundamental forces that govern the interactions at the organic-inorganic interface is therefore important for many areas of research and could lead to the design of new materials for optical, mechanical and biomedical applications. This paper demonstrates a single-molecule force spectroscopy technique that utilizes an AFM to measure the adhesion force between either peptides or amino acids and well-defined inorganic surfaces. This technique involves a protocol for attaching the biomolecule to the AFM tip through a covalent flexible linker and single-molecule force spectroscopy measurements by atomic force microscope. In addition, an analysis of these measurements is included.

Here we present a protocol to measure the force of interactions between a well-defined inorganic surface and either peptides or amino acids by single-molecule force spectroscopy measurements using an atomic force microscope (AFM). The information obtained from the measurement is important to better understand the peptide-inorganic material interphase.

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the inverse electron demand Diels-Alder (IEDDA) cycloaddition between a radiolabeled tetrazines and trans-cyclooctene (TCO) linked to a biomolecule has proven to be a highly effective bioorthogonal approach to imaging specific biological targets. Despite the fact that technetium-99m remains the most widely used isotope in diagnostic nuclear medicine, there is a scarcity of methods for preparing 99mTc-labeled tetrazines. Herein we report the preparation of a family of tridentate-chelate-tetrazine derivatives and their Tc(I) complexes. These hitherto unknown compounds were radiolabeled with 99mTc using a microwave-assisted method in 31% to 83% radiochemical yield. The products are stable in saline and PBS and react rapidly with TCO derivatives in vitro. Their in vivo pre-targeting abilities were demonstrated using a TCO-bisphosphonate (TCO-BP) derivative that localizes to regions of active bone metabolism or injury. In murine studies, the 99mTc-tetrazines showed high activity concentrations in knees and shoulder joints, which was not observed when experiments were performed in the absence of TCO-BP. The overall uptake in non-target organs and pharmacokinetics varied greatly depending on the nature of the linker and polarity of the chelate.

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Here, we describe a protocol for radiolabeling and in vivo testing of tridentate 99mTc(I) chelate-tetrazine derivatives for pre-targeting and bioorthogonal chemistry.

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the ...