Name: site directed spin labeling and epr spectroscopic studies of pentameric ligand-gated ion channels
Uploaded: 2016-07-04T15:00:00
Description: Watch this Scientific Journal Video about Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels at JoVE.com

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.

1. Site-Directed Mutagenesis and Cys Mutations
<ol>
	<li>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.
	<ol>
		<li>Mix 5 &#956;l of 10x reaction buffer, 1 &#956;l of 100 ng/&#956;l cysteine-less GLIC template DNA<s...

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...

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 functional characterization of these channel preparations and the mechanistic description of channel function.
Gating conformational changes are complex and occur over a 60 &#197; distance along the length of the channel and these transitions are extensively modulated by membrane lipids. In particular, negative lipids, cholesterol, and phospholipids have been shown to modulate the function of pLGIC8-11. While the precise role of these lipid constituents in channel function remains unknown, a complete molecular understanding of gating would require studying these channels in their native environment. Site-Directed Spin Labeling (SDSL) and Electron Paramagnetic Resonance (EPR) spectroscopy are the techniques of choice for studying protein dynamics in reconstituted systems. EPR spectroscopy is not limited by the molecular size (as is NMR) or the optical property of the sample (as is fluorescence spectroscopy), and thereby allows measurements of full-length constructs reconstituted in native lipid conditions. The technique is extremely sensitive and has relatively low sample requirements (in the pico-mole range). Both these aspects make the technique well suited for studying large membrane proteins that are difficult to express in over milligram quantities.
The use of EPR spectroscopy in combination with site-directed spin labeling was developed by Wayne Hubbell and colleagues, and has been adapted for studying a range of protein types.12-24 EPR data have been used to investigate secondary structures, changes in the protein conformation, membrane-insertion depths, and protein-protein/protein-ligand interactions.
The method involves cysteine substitution at positions of interest by site-directed mutagenesis. To ensure site-specific labeling, it is necessary to substitute native cysteines with another amino acid (e.g., serine) to create a cysteine-less template. By far, the most popular spin label is a thiol-specific MTSL: (1-oxyl-2,2,5,5-tetramethyl-&#916;3-pyrroline-3-methyl) methanethiosulfonate that attaches to the protein through a disulfide bond bridge. Due to its high specificity, relatively small size (slightly larger than tryptophan), and flexibility of the linker region, this spin label has been shown to have excellent reactivity even with a buried cysteine. Furthermore, to maximize reactivity, the labeling reaction of the protein is carried out in the detergent-solubilized form. After separation of the excess free spin-label by size exclusion chromatography, the protein is reconstituted into liposomes or bilayer-mimicking systems of defined lipid composition. In general, cysteine mutagenesis is well tolerated in most parts of the protein, and the relatively small size of the spin-probe causes minimal perturbation to the secondary and tertiary structures. To ensure that the modification retained wild type functions, the labeled and reconstituted channels can be studied by patch-clamp measurements.
The labeled-functional protein is then subjected to spectroscopic measurements, which essentially provide three main types of information:12,14,15,20,22,23,25-27 spin-probe dynamics by lineshape analysis; accessibility of the probe to paramagnetic relaxation agents; and distance distribution.27 EPR distances are measured by two different approaches. The first is based on the Continuous Wave (CW) technique, where spectral broadening arising from dipolar interactions between spin-labels (in the 8 - 20 &#197; distance range) is used to determine distance.28,29 The second is a pulsed-EPR method where distance measurements can be extended up to 70 &#197;.30-34 In Double Electron Electron Resonance (DEER), oscillations in the spin-echo amplitude are analyzed to determine distances and distance distributions. Here the spin echo is modulated at the frequency of the dipolar interaction. Together, these parameters are used to determine protein topology, secondary structural elements, and protein-conformational changes.

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		<tr>
			<td>Site-Directed Mutagenesis and Cys mutations</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10x PfuUltra HF reaction buffer</td>
			<td>Agilent Technologies</td>
			<td>600380-52</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPS</td>
			<td>New England BioLabs Inc&#8206;</td>
			<td>N0447L</td>
			<td>10mM each dNTP</td>
		</tr>
		<tr>
			<td>pfu Ultra DNA polymerase</td>
			<td>Agilent Technologies</td>
			<td>600380-51</td>
			<td>2.5 U/ul</td>
		</tr>
		<tr>
			<td>DPNI</td>
			<td>New England BioLabs Inc&#8206;</td>
			<td>R0176S</td>
			<td>20,000 U/ml</td>
		</tr>
		<tr>
			<td>XL10 GOLD</td>
			<td>Agilent Technologies</td>
			<td>200314</td>
			<td></td>
		</tr>
		<tr>
			<td>SOC media</td>
			<td>New England BioLabs Inc&#8206;</td>
			<td>B9020S</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Fisher Scientfic</td>
			<td>BP905</td>
			<td></td>
		</tr>
		<tr>
			<td>LB media</td>
			<td>Invitrogen</td>
			<td>127957084</td>
			<td></td>
		</tr>
		<tr>
			<td>Miniprep kit</td>
			<td>QIAGEN</td>
			<td>27106</td>
			<td></td>
		</tr>
		<tr>
			<td>C43 competent cells</td>
			<td>Lucigen</td>
			<td>60446</td>
			<td></td>
		</tr>
		<tr>
			<td>Expression and Purification</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Fisher Scientfic</td>
			<td>D16</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Fisher Bioreagents</td>
			<td>BP1421-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Amresco</td>
			<td>J850</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Bioreagents</td>
			<td>BP229</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Amresco</td>
			<td>0705</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Amresco</td>
			<td>0781</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG (isopropyl-thio-&#946;-galactoside)</td>
			<td>Gold Biotechnology</td>
			<td>I2481C25</td>
			<td></td>
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		<tr>
			<td>Trizma Base</td>
			<td>Sigma Life Science</td>
			<td>T1503</td>
			<td></td>
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			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
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			<td>DNase I</td>
			<td>Sigma Life Science</td>
			<td>DN25</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Amresco</td>
			<td>M145</td>
			<td></td>
		</tr>
		<tr>
			<td>Leupeptine</td>
			<td>Amresco</td>
			<td>J580</td>
			<td></td>
		</tr>
		<tr>
			<td>Pepstatin</td>
			<td>Amresco</td>
			<td>J583</td>
			<td></td>
		</tr>
		<tr>
			<td>DDM (n-Docecyl-&#946;-D-Maltopyranoside)</td>
			<td>Anatrace</td>
			<td>D310S</td>
			<td></td>
		</tr>
		<tr>
			<td>Amylose resin</td>
			<td>New England BioLabs Inc&#8206;</td>
			<td>E8021L</td>
			<td></td>
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		<tr>
			<td>TCEP</td>
			<td>Amresco</td>
			<td>K831</td>
			<td></td>
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		<tr>
			<td>EDTA</td>
			<td>Fisher Scientfic</td>
			<td>BP118</td>
			<td></td>
		</tr>
		<tr>
			<td>Maltose</td>
			<td>Acros Organics</td>
			<td>329915000</td>
			<td></td>
		</tr>
		<tr>
			<td>Superdex 200GL</td>
			<td>GE Healthcare</td>
			<td>17-5175-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Empty polypropylene Chromatography column</td>
			<td>BioRad</td>
			<td>731-1550</td>
			<td></td>
		</tr>
		<tr>
			<td>Site-Directed Spin Labeling</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MTSL (1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl) Methanethiosulfonate</td>
			<td>Toronto Reaserch chemicals Inc</td>
			<td>O873900</td>
			<td></td>
		</tr>
		<tr>
			<td>(1-acetoxy-2,2,5,5-tetramethyl-&#916;3-pyrroline-3-methyl) methanethiosulfonate</td>
			<td>Toronto Reaserch chemicals Inc</td>
			<td>A167900</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>J.T. Baker</td>
			<td>9224-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Reconstitution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Asolectin lipid</td>
			<td>Avanti polar lipids Inc</td>
			<td>541602C</td>
			<td></td>
		</tr>
		<tr>
			<td>Biobeads (Polystyrine beads)</td>
			<td>Bio Rad</td>
			<td>152-3920</td>
			<td></td>
		</tr>
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			<td>Methanol</td>
			<td>Fisher chemicals</td>
			<td>A413</td>
			<td></td>
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		<tr>
			<td>FRET</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein-maleimide</td>
			<td>ThermoFisher Scientific</td>
			<td>F-150</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylrhodamine-maleimide</td>
			<td>ThermoFisher Scientific</td>
			<td>T-6027</td>
			<td></td>
		</tr>
		<tr>
			<td>POPC</td>
			<td>Avanti polar lipids Inc</td>
			<td>850457C</td>
			<td></td>
		</tr>
		<tr>
			<td>POPG</td>
			<td>Avanti polar lipids Inc</td>
			<td>840457C</td>
			<td></td>
		</tr>
		<tr>
			<td>E.Coli polar lipid extract</td>
			<td>Avanti polar lipids Inc</td>
			<td>100600C</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Life Science</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>EPR measurement</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TPX plastic capillaries</td>
			<td>Bruker</td>
			<td>ER221</td>
			<td></td>
		</tr>
		<tr>
			<td>EDDA (Ethylenediamine-N, N&#39;-diacetic acid)</td>
			<td>Aldrich</td>
			<td>158186</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni(OH)2</td>
			<td>Aldrich</td>
			<td>283622</td>
			<td></td>
		</tr>
	</tbody>
</table>

site directed spin labeling and epr spectroscopic studies of pentameric ligand-gated ion channels

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.

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...

Watch this Scientific Journal Video about Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels at JoVE.com

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

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. ...

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