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.

Dette arbejde er blevet grundlagt af Deutsche Forschungsgemeinschaft (DFG) under tilskud CO822/2-1 (Emmy-Noether program) og CO822/3-1 til IC

1. fluorescens-baseret Screening af indarbejdelse effektivitetsfordele (figur 1)
<ol><li>Opretholde HEK293 celler i Dulbeccos modificerede Eagle Medium (DMEM; høje glukose, 4 mM glutamin, pyruvat) suppleret med 10% (v/v) føtal bovint serum (FBS), 100 U/mL penicillin og 100 µg/mL streptomycin ved 37 ° C, luftfugtighed 95% og 5% CO2.</li>
	<li>Frø celler dagen før Transfektion.<ol>
<li>Frigør cellerne i 5 min ved 37 ° C i 0,05% Trypsin/PBS suppleret med 0,5 mM EDTA. Brug 1...

Protokollen beskriver en simpel og pålidelig analyse til at vurdere effektiviteten af ortogonale par til indbygning af ncAAs i proteiner udtrykt i pattedyrsceller. Den største fordel ved denne metode i forbindelse med udbredte assays baseret på FACS er at det giver mulighed for samtidige forberedelse og måling af et større antal prøver, og indeholder data, der er let analyseret ved hjælp af en almindelig software. Tilgængeligheden af en medium-overførselshastighed metode til at analysere ortogonale par i pattedy...

Den genetiske indarbejdelse af kemiske sonder i proteiner er en kraftfuld metode til at lette undersøgelsen af strukturelle og dynamiske aspekter af protein funktion direkte i naturlig forbindelse med den levende celle. I dag, kan hundredvis af ikke-kanoniske aminosyrer (ncAAs) udstyret med de mest forskelligartede kemiske grupper være site-specifically indarbejdet i proteiner af biosyntesen1,2,3,4. Mellem dem, man finder lysfølsomt ncAAs såsom foto-overfladebehandlingsvæsker5, foto-bur6,7,8,9 og foto-omstillelig aminosyrer10, 11, aminosyrer forsynet med anstrengte alkener og Alkyner for katalysator-fri bioorthogonal kemi2,12,13,14,15,16 ,17, aminosyrer, der transporterer dansyl18, cumarin9,19og prodan20,21 fluorophores og aminosyrer udstyret med andre biofysiske sonder som godt som med post translationel ændringer1,2,3,4,22,23,24,25.
Den genetiske kodning af en ncAA er aktiveret af en dedikeret amino-acyl-tRNA-syntetase (AARS) parret til en beslægtet suppressor-tRNA, hvilke indlemmer ncAA som svar på en rav stop codon under den regelmæssige ribosomale syntese. ncAARS/tRNA par er konstrueret så den er ortogonale i værtsorganismen, dvs ikke cross-talk med de endogene par. Teknikken er veletableret både i prokaryote og eukaryote værter og let anvendelig til mammale celler. Par ncAA blandes i pattedyrceller er baseret på tre vigtigste ortogonale systemer: det tyrosyl system, der kombinerer TyrRS fra E. coli26 med en tyrosyl rav suppressor fra B. stearothermophilus27 (EF TyrRS /BstYam par), E. coli leucyl system (EFLeuRS/tRNALeuCUA par)6,18,28 , og archaeask pyrrolysyl system (PylRS/tRNA Pyl par)3, hvorved tRNAPyl er en naturlig rav suppressor. I almindelighed, er hver ncAA anerkendt af et specialiseret ncAARS. Afhængig af strukturen i ncAA opnås ncAARS via styret evolution af enten TyrRS, LeuRS eller PylRS, selv om nogle synthetases kan acceptere mere end én ncAA.
Ortogonale parret er importeret ind i cellerne ved blot at bruge et plasmid vektor. Mest almindelige og effektiv plasmider er bicistronic og indkode både for syntetase og tRNA danner ortogonale par29. En anden plasmid kodning for protein af rentebærende en rav codon på webstedet udpeget til ændring er co transfected. NcAA er simpelthen tilføjet til celle vækstmedium. Dog bruger forskellige specialiserede grupper ofte forskellige varianter af plasmid konstruktioner selv for indarbejdelse af den samme ncAA. Konstruerer forskellige arrangement af gener i vektor, type af syntetase, codon skik i syntetase gen, promoter skik, variant af tRNA og antallet af tRNA udtryk kassetter. Derudover kan indarbejdelse effektiviteten af forskellige ncAAs variere drastisk på grund af de forskellige synthetases, kvaliteten af tRNA, og andre faktorer30forskellige katalytisk effektivitet. Derfor er det vigtigt at have ved hånden en hurtig og pålidelig metode til at vurdere effektiviteten af en ortogonal par, både til at vælge den mest passende system for en ønskede program og til at udføre nogle optimering trin, der forbedrer samlede protein udtryk udbytter.
Vi har etableret en enkel og robust fluorescens-baseret analyse for at evaluere effektiviteten af ortogonale par29 (figur 1). I analysen, celler er co transfected med plasmid kodning for ortogonale parret sammen med en bicistronic reporter plasmid kodning både til den grønne fluorescerende proteiner forsynet med en rav stop codon på en eftergivende holdning (EGFPTAG) og den mCherry gen. Røde og grønne fluorescens af hele cellelysater læses i separate kanaler på en Pladelæser i en 96-brønd plade. Intensiteten af den grønne fluorescens direkte korrelerer med effektiviteten af rav undertrykkelse, der henviser til, at intensiteten af røde fluorescens giver et direkte estimat af størrelsen af den målte prøve og Transfektion effektivitet. Med hensyn til lignende assays baseret på fluorescens læse assisteret celle sortering (FACS) ud af31,32, analysen giver en øjeblikkelig og omfattende vurdering af protein udtryk i befolkningens hele cellen, som er mere repræsentant for sædvanlige forsøgsbetingelser, og byder på en lettere dataopsamling og behandling med standardsoftwaren. Samlet set er den største fordel af analysen, at et medie til et stort antal prøver kan analyseres parallelt. Ved hjælp af denne analyse, har vi screenet et rationelt designet bibliotek af suppressor-tRNAer til at forbedre effektiviteten af Pyl ortogonale system30. Dette arbejde beskriver forsøgsplan for at udføre denne analyse og vise eksempler på dens anvendelse, herunder optimering af ortogonale parret for indarbejdelse af den foto-crosslinking ncAA p-azido-L-fenylalanin (Amalie) og sammenligning af indarbejdelse effektiviteten af forskellige aminosyrer (figur 2).
I de seneste år, ncAA værktøjer har vist sig meget kraftfuld at undersøge strukturelle og funktionelle aspekter af G-protein koblede receptorer (GPCRs)33,34,35,36,37 , 38. i mennesker, GPCRs udgør en stor familie af membranreceptorer (800 medlemmer) og repræsenterer vigtigste mål for terapeutiske lægemidler. Direkte strukturel karakterisering af GPCRs er stadig udfordrende og supplerende biokemiske metoder er meget nødvendige for deres undersøgelse. Vi har været banebrydende brug af foto-crosslinking ncAAs kort GPCR overflader og oplev ligand bindende lommer34. Ved hjælp af vores optimeret system til Azi indbygning, indarbejdet vi systematisk Azi i hele domænet hele juxtamembrane af en GPCR direkte i live pattedyrsceller. Ved UV-bestråling danner Azi en meget reaktive nitrene arter, der kovalent fanger nabo molekyler. Når liganden føjes til systemet, fungerer Azi som en nærhed sonde til at afsløre, hvilke holdninger af receptor kommer tæt på den bundne ligand. På denne måde blev tilstanden bindende neuropeptid hormon Urocortin I (Ucn1) på klasse B GPCR corticotropin-releasing-faktor receptor type 1 (CRF1R)33 først afsløret. Sidst, har vi offentliggjort særskilt bindende mønstre af agonister og antagonister på samme receptor38. En lignende fremgangsmåde er blevet anvendt af andre til at afsløre orthosteric og allosteriske bindingssteder andre peptider og lille molekyle ligander på andre GPCRs39,40,41,42. Dette manuskript beskriver forsøgsplan anvendes i vores lab for foto-crosslinking kortlægning af GPCR overflader. Metoden er relativt hurtigt, ligetil og kræver ikke nogen særligt udstyr, således at det kan anvendes i standard biokemi labs. Vigtigere er, tilgangen giver et værdifuldt redskab, ikke kun til at identificere ligand bindingssteder hvor 3D strukturelle data er sparsomme, men også at supplere eksisterende i vitro data med oplysninger fra fuldt post-translationally modificerede receptorer i den fysiologiske miljø af den levende celle.
Den seneste udvikling af nye ncAAs indflydelse på de side kæde kemiske grupper egnet til ultrahurtig katalysator-fri bioorthogonal kemi har åbnet op for muligheden for at installere sidste generation fluorophores for super-opløsning imaging i proteiner direkte på de levende celler2,43. Sådanne kemiske ankre omfatter anstrengte cyclooctyne i SCOK14, bicyclo [6.1.0] nonyne i BCNK12,17, og trans-cyclooctenes i TCO * K13,15,17 blandt andre ncAAs husly en norbornene16,17,44 eller cyclopropene45,46 gruppe. Pladskrævende ncAAs i bioorthogonal kemi indgår med en variant af PylRS normalt betegnes som PylRSAF (med angivelse af mutation Y271A og Y349F i M. barkeri PylRS), samt af andre ad hoc- udviklet sig ncAARSs17 , 44. bioorthogonal ankre reagere med tetrazine reagenser47 via inverse elektron-demand Diels-elletræ cycloaddition at give høje mærkning udbytter inden for et par minutter43,48. Dog har anvendelsen af denne kraftfulde tilgang til label GPCRs udfordrende på grund af en lav samlede effektivitet af ortogonale ncAA indarbejdelse system. Bruge vores forbedrede Pyl system, har vi for nylig demonstreret højtydende indarbejdelse af sådanne aminosyrer i GPCRs og ultrahurtig GPCR mærkning på overfladen af levende pattedyrceller30. Mærket receptorer var stadig funktionel, som de fysiologisk internaliseret ved aktivering receptor med en agonist. Forsøgsplan for indarbejdelse af bioorthogonal ankre i GPCRs og de følgende mærkning trin er beskrevet her. Udstyre GPCRs med små lyse fluorophores er det første grundlæggende skridt mod studiet af GPCR strukturdynamik i den levende celle via avancerede mikroskopi-teknikker.

<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

Den genetiske indarbejdelse af ikke-kanoniske aminosyrer (ncAAs) via rav stop codon undertrykkelse er en kraftfuld teknik til at installere kunstige sonder og reaktive fraspaltning på proteiner direkte i den levende celle. Hver ncAA er indarbejdet i en dedikeret ortogonale suppressor-tRNA/amino-acyl-tRNA-syntetase (AARS) par, der importeres til værtsorganismen. Indarbejdelse effektiviteten af forskellige ncAAs kan variere, og være utilfredsstillende i nogle tilfælde. Ortogonale par kan forbedres ved at manipulere med enten de årlige aktivitetsrapporter eller tRNA. Styret evolution af tRNA eller AARS ved hjælp af store biblioteker og død/levende udvælgelsesmetoder er imidlertid ikke muligt i pattedyrsceller. Her præsenteres en letkøbt og robust fluorescens-baseret analyse til at evaluere effektiviteten af ortogonale par i pattedyrsceller. Analysen giver mulighed for screening snesevis til hundredvis af AARS/tRNA varianter med en moderat indsats og inden for en rimelig frist. Brug af denne analyse til at generere nye tRNAer, der væsentligt forbedrer effektiviteten af Pyrrolysin ortogonale system er beskrevet, samt anvendelse af ncAAs til studiet af G-protein koblede receptorer (GPCRs), som udfordrende objekter for ncAA mutagenese. Først, systematisk indarbejder en foto-crosslinking ncAA i hele den ekstracellulære overfladen af en receptor, bindingssteder af forskellige ligander på intakt receptoren er knyttet direkte i den levende celle. Anden, ved at indarbejde sidste generation ncAAs i en GPCR, ultrahurtig katalysator-fri receptor mærkning med et fluorescerende farvestof er demonstreret, som udnytter bioorthogonal stamme-forfremmet inverse Diels elletræ cycloaddition (SPIEDAC) på den levende celle. Som ncAAs kan anvendes generelt på et protein uafhængigt på sin størrelse, er metoden af almen interesse til en række applikationer. Derudover ncAA vedtægter kræver ikke nogen særligt udstyr og udføres nemt i standard biokemi labs.

Omridset af fluorescens assay er afbildet i figur 1. Analysen er ansat i tre programmer. I første omgang, er en række tRNA varianter til indbygning af Lys(Boc) af Pyl ortogonale parret screenet. Lys(BOC) er en sterically svarende til Pyl aminosyre. Da Pyl ikke er kommercielt tilgængelige, er Lys(Boc) almindeligt anvendt som en standard substrat for PylRS. De screenede tRNAer er baseret på tRNAPyl. Hver tRNA variant bærer mutationer af enkelt b...

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

En letkøbt fluorescens assay er præsenteret for at evaluere effektiviteten af amino-acyl-tRNA-syntetase/tRNA par indarbejde ikke-kanoniske amino-syre (ncAAs) i proteiner udtrykt i pattedyrsceller. Anvendelsen af ncAAs til at studere G-protein koblede receptorer (GPCRs) er beskrevet, herunder foto-crosslinking kortlægning af bindende websteder og bioorthogonal GPCR mærkning på levende celler.

Optimering af den genetiske indarbejdelse af kemiske sonder ind i GPCRs til foto-crosslinking kortlægning og Bioorthogonal kemi i Live pattedyrceller

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.

Research

JoVE Journal

Chemistry

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