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Química

Webstedet Instrueret Spin Mærkning og EPR spektroskopiske Undersøgelser af pentamere ligand ionkanaler

Published: July 04, 2016
doi:
10.3791/54127
, ,
1Department of Physiology and Biophysics,Case Western Reserve University, 2School of Medicine,Case Western Reserve University

Summary

This article describes methods for site-directed spin labeling and reconstitution of pentameric ligand-gated channels for Electron Paramagnetic Resonance studies. This protocol can be adapted for any membrane protein. The reconstitution method described here can also be used for patch-clamp measurements of macroscopic and single-channel currents in a defined lipid system.

Abstract

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. Fundamental to these transitions is the intrinsic flexibility of the protein, which is critically modulated by membrane lipid-composition. To better understand the structural basis of channel function, it is necessary to study protein dynamics in a physiological membrane environment. Electron Paramagnetic Resonance (EPR) spectroscopy is an important tool to characterize conformational transitions between functional states. In comparison to NMR and X-ray crystallography, the information obtained from EPR is intrinsically of lower resolution. However, unlike in other techniques, in EPR there is no upper-limit to the molecular weight of the protein, the sample requirements are significantly lower, and more importantly the protein is not constrained by the crystal lattice forces. Therefore, EPR is uniquely suited for studying large protein complexes and proteins in reconstituted systems. In this article, we will discuss general protocols for site-directed spin labeling and membrane reconstitution using a prokaryotic proton-gated pentameric Ligand-Gated Ion Channel (pLGIC) from Gloeobacter violaceus (GLIC) as an example. A combination of steady-state Continuous Wave (CW) and Pulsed (Double Electron Electron Resonance-DEER) EPR approaches will be described that will enable a complete quantitative characterization of channel dynamics.

Introduction

I det seneste årti har den strukturelle forståelse af pentamere ligand-gatede ionkanaler (pLGIC) vokset med stormskridt på grund massevis af højopløselige strukturer af flere medlemmer af familien. Vigtige faktorer, der førte til de nuværende fremskridt inden omfatter, opdagelsen af prokaryote pLGIC kanaler, 1-3 store fremskridt inden eukaryote membranprotein udtryk, 4-6 og enorme gennembrud i struktur beslutsomhed tilgange. 7. Disse strukturer giver en klar enighed om den overordnede beskyttelse af den tredimensionale arkitektur af pLGIC. Men to store områder, der synes at trail bag er den funktionelle karakterisering af disse kanal præparater og mekanistisk beskrivelse af kanal funktion.

Gating konformationelle ændringer er komplekse og forekommer over en 60 Å afstand langs længden af ​​kanalen og disse overgange er udførligt moduleres vedmembranlipider. Især har negative lipider, cholesterol og phospholipider vist sig at modulere funktionen af pLGIC 8-11. Mens den præcise rolle af disse lipid bestanddele i kanal funktion forbliver ukendt, ville en komplet molekylær forståelse af gating kræver at studere disse kanaler i deres oprindelige miljø. Site-directed Spin Mærkning (SDSL) og elektronspinresonans (EPR) spektroskopi er de teknikker til valg for at studere protein dynamik i rekonstituerede systemer. EPR-spektroskopi er ikke begrænset af den molekylære størrelse (som er NMR) eller den optiske egenskab af prøven (som er fluorescensspektroskopi), og tillader derved målinger af fuld længde konstruktioner rekonstitueret i native lipid forhold. Teknikken er yderst følsom og har krav relativt lave prøve (i pico-mol interval). Begge disse aspekter gør teknikken velegnet til at studere store membranproteiner, der er vanskelige at udtrykke i løbet milligrammængder.

Anvendelsen af EPR-spektroskopi i kombination med steddirigeret centrifugering mærkning blev udviklet af Wayne Hubbell og kolleger, og er blevet tilpasset til at studere en række protein-typer. 12-24 EPR data er blevet anvendt til at undersøge sekundære strukturer, ændringer i proteinet konformation, membran-indsættelse dybder, og protein-protein / protein-ligand-interaktioner.

Fremgangsmåden involverer cystein substitution ved positionerne af interesse ved site-directed mutagenese. At sikre stedspecifik mærkning, er det nødvendigt at erstatte native cysteiner med en anden aminosyre (f.eks., Serin) at skabe en cystein-mindre skabelon. Langt den mest populære spin-mærket er et thiol-specifik MTSL: (1-oxy 2,2,5,5-tetramethyl-Δ3-pyrrolin-3-methyl) methanethiosulfonate der tillægger proteinet gennem en disulfidbinding bro. På grund af sin høje specificitet, relativt lille størrelse (lidt større end tryptophan), og flexiheden for linker-regionen, har denne spinmærkning vist sig at have fremragende reaktivitet selv med en nedgravet cystein. Endvidere at maksimere reaktiviteten reaktionen af ​​proteinet mærkning udføres i detergent-solubiliserede formular. Efter separation af overskydende frie spin-mærke ved gelpermeationskromatografi proteinet rekonstitueres til liposomer eller dobbeltlag-efterlignende systemer med defineret lipidsammensætning. Generelt er cystein mutagenese veltolereret i de fleste dele af proteinet, og den relativt lille størrelse af spin-sonde forårsager minimal forstyrrelse til de sekundære og tertiære strukturer. At sikre, at modifikationen bevaret vildtype-funktioner, kan de mærkede og rekonstituerede kanaler studeres ved patch-clamp målinger.

Det mærkede-funktionelle protein udsættes derefter for spektroskopiske målinger, som i det væsentlige giver tre hovedtyper af oplysninger: 12,14,15,20,22,23,25-27 spin-probe dynamik ved lineshabe-analyse; tilgængelighed af proben til paramagnetiske afslapning midler; og afstand distribution. 27 EPR afstande måles ved to forskellige fremgangsmåder. Den første er baseret på den Continuous Wave (CW) -teknik, hvor spektral udbredning følge af dipolære vekselvirkninger mellem spin-etiketter (i 8 – 20 A afstandsområde). Anvendes til at bestemme afstanden 28,29 Den anden er en pulseret-EPR metode, hvor afstandsmålinger kan forlænges op til 70 Å. 30-34 i Double Electron Electron Resonance (DEER), er svingninger i spin-ekko amplitude analyseret for at bestemme afstande og afstande distributioner. Her spin ekko moduleres ved frekvensen af ​​den dipolære interaktion. Tilsammen er disse parametre anvendes til at bestemme protein topologi, sekundære strukturelementer, og protein-konformationelle ændringer.

Protocol

1. steddirigeret mutagenese og Cys Mutationer Kloning og mutagenese BEMÆRK: GLIC vildtype (wt) 35 har en enkelt-native cystein (C27), der er muteret til serin at skabe en cystein-mindre baggrund. Cystein indføres mutationer på cystein-mindre baggrund ved site-directed mutagenese ved anvendelse af primere, der bærer en cysteinkodon ved den ønskede position 36. Bland 5 eliter 10X reaktionsbuffer, 1 pi 100 ng / pl cystein-mindre GLIC template DNA 35,…

Representative Results

Biokemisk karakterisering af Spin-mærket GLIC mutanter Efter den ovenfor beskrevne protokol ville typisk give GLIC-MBP-fusionsproteinet i området på 10 – 12 mg / l kultur. Selv om denne værdi kan variere på tværs af forskellige mutanter, især for positioner begravet i proteinet, kan udbyttet blive væsentligt kompromitteret. I disse tilfælde kan de mængder kultur kræver opskalering. Spaltningen af ?…

Discussion

EPR-spektroskopi har vist sig at være en enestående strukturel tilgang kvantificering konformationelle ændringer i membranproteiner i en nær-native miljø. Denne tilgang giver os et kig ind i de molekylære detaljer af protein dynamik, der er skjult i høj opløsning strukturer fra røntgenkrystallografi og Cryo-elektronmikroskopi. Men det er vigtigt at overveje de tekniske begrænsninger ved denne fremgangsmåde, der kan påvirke den generelle anvendelighed til andre systemer, og også at huske på de potentielle e…

Declarações

The authors have nothing to disclose.

Acknowledgements

Vi er meget taknemmelige for de nuværende og tidligere medlemmer af Chakrapani laboratorium for kritisk læsning og kommentarer til manuskriptet. Dette arbejde blev støttet af National Institutes of Health tilskud (1R01GM108921) og American Heart Association (NCRP Scientist Development Grant 12SDG12070069) og SC.

Materials

Site-Directed Mutagenesis and Cys mutations
10x PfuUltra HF reaction buffer Agilent Technologies 600380-52
dNTPS New England BioLabs Inc‎ N0447L 10mM each dNTP
pfu Ultra DNA polymerase Agilent Technologies 600380-51 2.5 U/ul
DPNI New England BioLabs Inc‎ R0176S 20,000 U/ml
XL10 GOLD Agilent Technologies 200314
SOC media New England BioLabs Inc‎ B9020S
Kanamycin Fisher Scientfic BP905
LB media Invitrogen 127957084
Miniprep kit QIAGEN 27106
C43 competent cells Lucigen 60446
Expression and Purification
Glucose Fisher Scientfic D16
Tryptone Fisher Bioreagents BP1421-500
Yeast extract Amresco J850
Glycerol Fisher Bioreagents BP229
K2HPO4 Amresco 0705
KH2PO4 Amresco 0781
IPTG (isopropyl-thio-β-galactoside) Gold Biotechnology I2481C25
Trizma Base Sigma Life Science T1503
NaCl Sigma-Aldrich S7653
DNase I Sigma Life Science DN25
PMSF Amresco M145
Leupeptine Amresco J580
Pepstatin Amresco J583
DDM (n-Docecyl-β-D-Maltopyranoside) Anatrace D310S
Amylose resin New England BioLabs Inc‎ E8021L
TCEP Amresco K831
EDTA Fisher Scientfic BP118
Maltose Acros Organics 329915000
Superdex 200GL GE Healthcare 17-5175-01
Empty polypropylene Chromatography column BioRad 731-1550
Site-Directed Spin Labeling
MTSL (1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl) Methanethiosulfonate Toronto Reaserch chemicals Inc O873900
(1-acetoxy-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl) methanethiosulfonate Toronto Reaserch chemicals Inc A167900
DMSO J.T. Baker 9224-01
Reconstitution
Asolectin lipid Avanti polar lipids Inc 541602C
Biobeads (Polystyrine beads) Bio Rad 152-3920
Methanol Fisher chemicals A413
FRET
Fluorescein-maleimide ThermoFisher Scientific F-150
Tetramethylrhodamine-maleimide ThermoFisher Scientific T-6027
POPC Avanti polar lipids Inc 850457C
POPG Avanti polar lipids Inc 840457C
E.Coli polar lipid extract Avanti polar lipids Inc 100600C
HEPES Sigma Life Science H3375
EPR measurement
TPX plastic capillaries Bruker ER221
EDDA (Ethylenediamine-N, N'-diacetic acid) Aldrich 158186
Ni(OH)2 Aldrich 283622
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Tags

Keywords: Site Directed Spin LabelingEPR SpectroscopyPentameric Ligand-gated Ion ChannelsMembrane Protein DynamicsGLICSite-directed MutagenesisProtein PurificationCell LysisMembrane Protein Reconstitution
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Basak, S., Chatterjee, S., Chakrapani, S. Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels. J. Vis. Exp. (113), e54127, doi:10.3791/54127 (2016).
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