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

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

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 ....

Protocol

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 DNA³⁵, 0.5 μl each of 40 μM forward and reverse primer³⁶, 1 μl of dNTPs (100 mM), and 41 μl of deionized water. Pipette the sample a few times to mix ....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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	10 mM 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
K2HPO4	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 200 GL	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