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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Chemistry

Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

Published: July 4th, 2016

DOI:

10.3791/54127

1Department of Physiology and Biophysics, Case Western Reserve University, 2School of Medicine, Case Western Reserve University

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.

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.

Over the last decade, the structural understanding of pentameric ligand-gated ion channels (pLGIC) has grown in leaps and bounds, owing to multitudes of high-resolution structures of several members of the family. Key factors that led to the current advancements in the field include, the discovery of prokaryotic pLGIC channels,1-3 major progresses in eukaryotic membrane protein expression,4-6 and tremendous breakthroughs in structure determination approaches.7 These structures provide a clear consensus on the overall conservation of the three-dimensional architecture of pLGIC. However, two major areas that seem to trail behind are the ....

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1. Site-Directed Mutagenesis and Cys Mutations

  1. Cloning and Mutagenesis
    NOTE: GLIC wild type (wt) 35 has a single-native cysteine (C27), which is mutated to serine to create a cysteine-less background. Cysteine mutations are introduced on the cysteine-less background by site-directed mutagenesis using primers that carry a cysteine codon at the desired position36.
    1. Mix 5 μl of 10x reaction buffer, 1 μl of 100 ng/μl cysteine-less GLIC template DNA

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Biochemical Characterization of Spin-labeled GLIC Mutants

Following the above described protocol would typically yield GLIC-MBP fusion protein in the range of 10 - 12 mg/L of culture. Although this value may vary across different mutants, particularly for positions buried within the protein, the yield may be significantly compromised. In these cases, the culture volumes may require scaling up. The cleavage of th.......

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EPR spectroscopy has proven to be an unparalleled structural approach in quantifying conformational changes in membrane proteins in a near-native environment. This approach allows us a peek into the molecular details of protein dynamics that are obscured in high-resolution structures from X-ray crystallography and Cryo-electron microscopy. However, it is important to consider the technical limitations of this approach that may affect the general applicability to other systems and also to keep in mind the potential experi.......

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We are very grateful to the current and former members of the Chakrapani lab for critical reading and comments on the manuscript. This work was supported by the National Institutes of Health grant (1R01GM108921) and the American Heart Association (NCRP Scientist Development Grant 12SDG12070069) and to SC.

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Name Company Catalog Number Comments
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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