We are very grateful to Dr. H. Mchaourab for providing access to the EPR and DEER spectrometers and Dr. T. Rosenbaum for providing the full-length cysteine-less TRPV1 plasmid.

1. TRPV1 Mutagenesis
Note: A minimal TRPV1 construct for spectroscopic analysis26 was built from the full-length cysteine-less channel TRPV127 using the polymerase chain reaction (PCR) method (Figure 1). This cysteine-less minimal TRPV1 construct (referred to as eTRPV1 hereafter) consists of residues 110 - 603 and 627 - 764. eTRPV1 was cloned in pMO (a pcDNA3.1-based vector) for functional analysis and in a recombinant...

The authors have nothing to disclose.

Current technologies for expression and purification of mammalian membrane proteins have made it possible to obtain sufficient amounts of protein for spectroscopic studies14,15,16,42. Here, we have adapted these technologies to express, purify, reconstitute, and perform spectroscopic analyses in TRPV1.
Among the critical steps in the protocol, below are the ones that...

With recent advances in single-particle cryo-electron microscopy (cryo-EM), mammalian ion channel structures have been obtained at an extraordinary rate. Particularly, structural studies of polymodal ion channels, such as the transient receptor potential vanilloid 1 (TRPV1), have provided further understanding of its activation mechanisms1,2,3,4,5. However, dynamic information about ion channels embedded in a membrane environment is required to understand their polymodal gating and drug-binding mechanisms.
Electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) spectroscopies have provided some of the most definitive mechanistic models for ion channels6,7,8,9,10,11,12,13. These approaches have been mainly restricted to the examination of prokaryotic and archeal ion channels that yield a large amount of detergent-purified proteins when overexpressed in bacteria. With the development of eukaryotic membrane proteins production in insect and mammalian cells for functional and structural characterization14,15,16, it is now possible to obtain biochemical amounts of detergent-purified proteins for spectroscopic studies.
The EPR and DEER signals arise from a&#160;paramagneticspin label (SL) (i.e., methanethiosulfonate) attached to a single-cysteine residue in the protein. The spin-labels report three types of structural information: motion, accessibilities, and distances. This information allows determining whether residues are buried within the protein or are exposed to the membrane or aqueous environment in the apo and ligand-bound states13,17,18,19. In the context of a high-resolution structure (when available), the EPR and DEER data provide a collection of constraints for deriving dynamic models in their native environment while monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Moreover, flexible regions that might be difficult to determine by X-ray crystallography or cryo-EM could be obtained by using these environmental data sets to assign secondary structures as well as location within the protein20. Cryo-EM structures obtained in lipid nanodiscs provided valuable information about the gating of ion channels3,21,22,23,24,25; however, spectroscopic approaches could provide dynamic information from conformational states (e.g., thermal changes) that might be difficult to determine using cryo-EM.
Many difficulties must be overcome to implement EPR and DEER, including lack of protein function when removing all cysteine residues (especially abundant in mammalian channels), low protein yield, protein instability during purification and after spin labeling, and protein aggregation in detergent or liposomes. Here, we have designed protocols to overcome these critical barriers and have obtained DEER and EPR spectra information for a mammalian sensory receptor. The purpose here is to describe methodologies for the expression, purification, labeling, and reconstitution of a functional minimal cysteine-less rat TRPV1 (eTRPV1) construct for spectroscopic analyses. This methodology is appropriate for those membrane proteins that keep their function despite the removal of cysteine residues or that contain cysteine forming disulfide-bonds. This collection of protocols could be adapted for the spectroscopic analysis of other mammalian ion channels.

<table border="0" cellpadding="0" cellspacing="0" style="width:1048px;" width="1047">
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			<td height="40" style="height:40px;width:339px;">QuikChange Lightning Site-Directed Mutagenesis Kit</td>
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			<td height="40" style="height:40px;">Collagenase Type 1</td>
			<td>Worthington-Biochem</td>
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			<td height="40" style="height:40px;">DDM (n-Docecyl-B-D-Maltopyranoside)</td>
			<td>Anatrace</td>
			<td>D310S</td>
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		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">High glucose medium (Dulbecco&#8217;s Modified Eagle&#8217;s Medium)</td>
			<td>Sigma</td>
			<td>D0572&#160;</td>
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			<td height="40" style="height:40px;">Disposable PD-10 Desalting Columns</td>
			<td>GE Healthcare</td>
			<td>45-000-148</td>
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			<td height="40" style="height:40px;">EGTA</td>
			<td>Fisher Scientific</td>
			<td>O2783</td>
			<td></td>
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			<td height="40" style="height:40px;">Fetal Bovine Serum</td>
			<td>Invitrogen Life Technologies</td>
			<td>10082-147</td>
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			<td height="40" style="height:40px;">Fluo-4 AM</td>
			<td>Life Technologies</td>
			<td>F-14201</td>
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			<td height="40" style="height:40px;">GenCatch Plus Plasmid DNA Mini-Prep Kit</td>
			<td>Epoch Life Science, Inc</td>
			<td>2160250</td>
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			<td height="40" style="height:40px;">GenCatch PCR Cleanup Kit</td>
			<td>Epoch Life Science, Inc</td>
			<td>2360050</td>
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			<td height="40" style="height:40px;">Gentamicin Sulfate</td>
			<td>Lonza</td>
			<td>17-518Z</td>
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		<tr height="40">
			<td height="40" style="height:40px;">Glass capillary (25 &#181;l)</td>
			<td>VWR</td>
			<td>53432-761</td>
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			<td height="40" style="height:40px;">Glass Flask 2800 mL</td>
			<td>Pyrex USA</td>
			<td>4423-2XL</td>
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			<td height="40" style="height:40px;">Glycerol</td>
			<td>Fisher BioReagents</td>
			<td>BP229</td>
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			<td height="40" style="height:40px;">HEK293S GnTl-</td>
			<td>ATCC</td>
			<td>CRL-3022</td>
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			<td height="40" style="height:40px;">HEPES</td>
			<td>Sigma&#160;</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">IPTG (isopropyl-thio-B-galactoside)</td>
			<td>GoldBio.com</td>
			<td>I2481C25</td>
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		<tr height="40">
			<td height="40" style="height:40px;">Kanamycin Sulfate</td>
			<td>Fisher Scientific</td>
			<td>BP906-5</td>
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		<tr height="40">
			<td height="40" style="height:40px;">KCl</td>
			<td>Fisher Chemical</td>
			<td>P217</td>
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			<td height="40" style="height:40px;">LB Broth, Miller</td>
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			<td>BP1426</td>
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			<td height="40" style="height:40px;">Leupeptin Hemisulfate</td>
			<td>GoldBio.com</td>
			<td>L-010-5</td>
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		<tr height="40">
			<td height="40" style="height:40px;">Lipofectamine 2000</td>
			<td>Invitrogen Life Technologies</td>
			<td>11668-019</td>
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		<tr height="40">
			<td height="40" style="height:40px;">MgCl2 6H2O</td>
			<td>Fisher Scientific</td>
			<td>BP214</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;">MgSO4 7H2O</td>
			<td>Fisher Scientific</td>
			<td>BP213</td>
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			<td height="40" style="height:40px;">mMESSAGE mMACHINE T7 Kit</td>
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			<td>AM1344</td>
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			<td height="40" style="height:40px;">MOPS</td>
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			<td>BP2936</td>
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		<tr height="40">
			<td height="40" style="height:40px;">MTSL (1-Oxyl-2,2,5,5-tetramethylpyrrolidin-3-yl) Methyl Methanethiosulfonate</td>
			<td>Toronto Research Chemicals, Inc</td>
			<td>O873900</td>
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		<tr height="40">
			<td height="40" style="height:40px;">NaCl</td>
			<td>Fisher Chemical</td>
			<td>S271</td>
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			<td height="40" style="height:40px;">Opti-MEM</td>
			<td>Life Technologies</td>
			<td>31985-062</td>
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		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Pepstatin A</td>
			<td>GoldBio.com</td>
			<td>P-020-5</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Pluronic Acid F-127 (20%)</td>
			<td>PromoKine&#160;&#160;</td>
			<td>CA707-59004</td>
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		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PMSF</td>
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			<td>P4170</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;">Poly-L-lysine Solution</td>
			<td>Sigma-Aldrich</td>
			<td>P4707</td>
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		<tr height="40">
			<td height="40" style="height:40px;">Rneasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74104</td>
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		<tr height="40">
			<td height="40" style="height:40px;">Sealed capillary</td>
			<td>VitroCom</td>
			<td>special order</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;">SF-900 II SFM (insect cell medium)</td>
			<td>Gibco, Life Technologies</td>
			<td>10902-088</td>
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		<tr height="40">
			<td height="40" style="height:40px;">Sf9 Cells (SFM Adapted)</td>
			<td>Invitrogen Life Technologies</td>
			<td>11496-015</td>
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			<td height="40" style="height:40px;">Soybean Polar Lipid Extract</td>
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			<td>541602C</td>
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			<td height="40" style="height:40px;">Sucrose</td>
			<td>Fisher Scientific</td>
			<td>S25590</td>
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		<tr height="40">
			<td height="40" style="height:40px;">Superose 6 Increase 10/300 GL</td>
			<td>GE Healthcare</td>
			<td>29091596</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">TCEP HCl</td>
			<td>GoldBio.com</td>
			<td>TCEP1</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tetracyclin Hydrochloride</td>
			<td>Fisher Scientific</td>
			<td>BP912-100</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tris Base</td>
			<td>Fisher BioReagents</td>
			<td>BP152</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tryptone</td>
			<td>Difco</td>
			<td>0123-01</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">X-gal</td>
			<td>GoldBio.com</td>
			<td>X4281C</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Xenopus oocytes</td>
			<td>Nasco</td>
			<td>LM00935M</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">XL1 - Blue Competent Cells</td>
			<td>Agilent Technologies, Inc</td>
			<td>200249</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Yeast Extract</td>
			<td>Difco</td>
			<td>0127-01-7</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:339px;">Econo-Pack chromatography column</td>
			<td style="width:292px;">Bio-Rad</td>
			<td>7321010</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini-PROTEAN TGX Stain-Free Precast Gels</td>
			<td style="width:292px;">Bio-Rad</td>
			<td>17000436</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">pFastBac1 Expression Vector</td>
			<td style="width:292px;">Invitrogen Life Technologies</td>
			<td style="width:251px;">10360-014</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">DH10Bac Competent Cells</td>
			<td style="width:292px;">Invitrogen Life Technologies</td>
			<td style="width:251px;">10361-012</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Critiseal capillary tube sealant</td>
			<td>Leica Microsystems</td>
			<td>02-676-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ABI Model 3130XL Genetic Analyzers</td>
			<td>Applied Biosystems</td>
			<td>4359571</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transfer pipete</td>
			<td>Fishebrand</td>
			<td>13-711-9AM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nanoject II</td>
			<td>Drummond Scientific</td>
			<td>3-000-204</td>
			<td></td>
		</tr>
	</tbody>
</table>

purification and reconstitution of trpv1 for spectroscopic analysis

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.

Functional Characterization of the Minimal Cysteine-less TRPV1 Construct (eTRPV1) and Single-cysteine Mutants
The first step toward spectroscopic studies is to engineer and characterize cysteine-less protein constructs (Figure 2A) that are functional and yield biochemical amounts of proteins. eTRPV1 is functional as determined by Ca2+ imaging and TEVC (Figure 2B...

Watch this Scientific Journal Video about Purification and Reconstitution of TRPV1 for Spectroscopic Analysis at JoVE.com

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

This article describes specific methods to obtain biochemical quantities of detergent-solubilized TRPV1 for spectroscopic analysis. The combined protocols provide biochemical and biophysical tools that can be adapted to facilitate structural and functional studies for mammalian ion channels in a membrane-controlled environment.

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

Here in the lab, we think that using a spectroscopic approach can help answering key questions in the transient receptor potential ion channel family. Such as determining the dynamic informational changes associated with polymodal activation. The main advantage of this protocol is that allow milligram of pure and functional remaining ion channel for electron paramagnetic resonance and double electron electron resonance epitroscopy.This method can provide insight into the conformational changes compatible with thermal and light independent gating of 3B1. It can also be applied to other trip channels, like 3B2, for example. Begin this procedure with design mutagenesis primers with online tools.Prepare the PCR reaction as described in the text protocol and spin the mixture. Place the tubes in the thermocycler and perform PCR mutagenesis using the PCR parameters listed in the text protocol. Then add two microliters of DPN1 enzyme to the reaction and mix.Incubate the reactions at 37 degrees Celsius for 30 minutes. Next, perform the transformation by adding 75 microliters of thawed E.Coli competent cells and 10 microliters of DPN1 treated PCR mixture to a pre-cooled 14 milliliter sterile tube. After mixing gently, incubate reaction on ice for 30 minutes.Maintain the tube at 42 degrees Celsius for 45 seconds and then immediately place it on ice for two minutes. Plate the mixture on an LB agar plate containing carbenicillin, and incubate overnight at 37 degrees Celsius. After harvesting single colonies and preparing overnights as described in the text protocol, spin down overnight cultures at 8000 times G for 10 minutes.Then, extract the DNA following the instructions of commercial available mini preparation kits. To generate the recombinant bacmid, transform 100 microliters of DH10bac E.Coli competent cells with 7.5 nanograms of the recombinant donor vector. Vectors contain the functional minimal cysteine-less rat TRPV1, abbreviated eTRPV1, and or single cysteine mutants.After seeding and incubating cells as described in the text protocol, select white colonies that are two millimeters in diameter since blue colonies lack the insert of interest. Use a stereo microscope to verify that white colonies do not contain any blue spots. Harvest at least three single colonies and transfer them into a 14 milliliter steril tube containing four milliliters supplemented LB media.Incubate the cells overnight at 37 degrees Celsius in 250 RPM. Take 1.5 milliliters of the overnight cultures and isolate recombinant bacmid DNA. To generate the recombinant baculovirus, plate one million SF9 cells in two millimeters of insect cell media.Allow the cells to attach for at least 45 minutes. After washing once with one milliliter of fresh medium, remove the wash medium and add DNA transfection reagent mixture to the cells. Incubate the cells at 27 degrees Celsius for five hours without agitation.Following incubation, remove the transfection mixture and replace with two milliliters of insect cell media containing 0.5%fetal bovine serum. Then, incubate the cells at 27 degrees Celsius for five days. Transfer the cell culture media into a 15 milliliter centrifuge tube.Isolate the first passage of viral stock by spinning the tube at 8000 times G for 15 minutes at 4 degrees Celsius. Transfer the supernatant to a clean 15 milliliter centrifuge tube and immediately store it at four degrees Celsius. Of the second generation of viral stock, pipette a 25 milliliter suspension culture of insect cell media at one million SF9 cells per milliliter containing 2%FBS into a 125 milliliter flask.Add 25 microliters of the P1 virus to the 25 milliliter culture. Then, incubate the culture at 27 degrees Celsius for 72 hours. To harvest the P2 viral stock transfer the culture to a new 50 milliliter tube and centrifuge it at 8000 times G for 15 minutes at four degrees Celsius.Transfer the supernatant to a new 50 milliliter tube and immediately store it at four degrees Celsius. Add one milliliter of P2 virus stock to the one liter culture and incubate it at 27 degrees Celsius for 72 hours. Harvest the one liter insect cell suspension culture by spinning at 4500 G for 20 minutes at four degrees Celsius.Weigh the cell pellet. It is expected to be around six to nine grams. Resuspend the cell pellet in 25 milliliters of ice cold buffer A in the presence of protease inhibitors.Disaggregate cell clumps to obtain a homogenous suspension and rotate at four degrees Celsius for 20 minutes. Break the cells using a manual or a high-pressure homogenizer. After removing the cell debris by centrifugation at 8000 times G for 20 minutes a four degrees Celsius, collect the supernatant and centrifuge again at 100, 000 times G for 30 minutes at four degrees Celsius.Following centrifugation, discard the supernatant and suspend the membrane pellet in a total volume of 20 milliliters of ice cold buffer B supplemented with protease inhibitors. Aliquot 20 milliliters of membrane suspension to 50 milliliter tubes. To perform the protein purification add three milliliters of DDM per tube.Rotate the tubes for two hours at four degrees Celsius. After centrifuging the sample at 100, 000 times G for 30 minutes at four degrees Celsius, collect the supernatant and add one milliliter of clean, wet amylose resin. Rotate the mixture for two hours at four degrees Celsius.Then load the protein amylose mixture in gravity flow chromatography columns. Wash the protein bound amylose resin with 10 times the bed volume of ice cold buffer C, followed by 10 bed volumes of ice cold buffer D.Elute eTRPV1 protein with 0.5 milliliter fractions up to five milliliters of ice cold degassed buffer D supplemented with 20 millimolar maltose. Perform the washes and elution at four degrees Celsius.Using a 100 kilodalton cutoff centrifugal filter unit, concentrate the eTRPV1 containing fractions to between two and 2.5 milligrams per milliliter for labeling. Next, add a ten-fold molar excess of MTSSL spin label from a 100 millimolar stock solution in DMSO to the concentrated protein. Repeat this twice every 30 minutes.Keep the reaction in the dark at room temperature for one hour and 30 minutes, followed by overnight incubation at four degrees Celsius. Then, load the spin label to eTRPV1 on a size-exclusion chromatography column equilibrated in buffer E, controlled by a fast protein liquid chromatography system. Collect the fractions containing eTRPV1 tetramer.Dry 10 milligrams of asolectin using a rotary evaporator under vacuum for one hour at 40 degrees Celsius. To make asolectin liposomes, add one milliliter of buffer F and sonicate the mixture for 15 minutes or until the sample is homogenous. Destabilize the liposomes by adding DDM to a final concentration of two millimolar and incubate for 30 minutes at room temperature.After adjusting the mixture to the DDM critical mycel concentration, as detailed in the text protocol, double the volume of the protein liposome mixture with buffer F.Remove the detergent by sequentially adding three aliquots of non-polar polystyrene absorbent beads at one hour intervals with gentle agitation at room temperature. Load the mixture onto a column filter to remove the beads and transfer the proteoliposome mixture to an ultra-centrifuge tube. Centrifuge the samples at 100, 000 times G for one hour at four degrees Celsius.Discard the supernatant and resuspend the pellet in 30 microliters of buffer F, and then perform spectroscopic measurements and analysis. The first checkpoint is the functional analysis of single-cysteine mutants before undertaking expression and purification protocols. Here, hek293 cells expressing wild type TRPV1, eTRPV1, and mutants were analyzed for capsaicin evoked responses using florescence calcium imaging.The second checkpoint is the biochemical characterization of spin labeled single-cysteine mutants through size-exclusion chromatography before proceeding with reconstitution and spectroscopic analysis. The third checkpoint is the functional characterization of reconstituted spin labeled single-cysteine mutants before undertaking spectroscopic analysis. Current voltage relationships determined by two electrode voltage clamp from Xenopus oocytes micro-injected with proteoliposomes containing spin labeled A702C were challenged with pH5 and blocked by capsazepine.Finally, it is possible to obtain the mobilities of spin labeled eTRPV1 mutants determined by continuous wave EPR. The first derivative spectra are shown for spin labeled cysteine mutants in DDM solution, and reconstituted in asolectin liposomes. Distances and mobilities of spin labeled eTRPV1 mutants in detergent were also determined by DEER.DEER echo and distance distribution of spin labeled mutant E651C is shown. After watching this video, you should have a good understanding of how to express, beautify, and reconstitute a mammalian ion channel for spectroscopic analysis. Generally, individuals new to this method will struggle because of the lack of protein function when removing cysteine residues, low protein yield, and protein instability either in purification and after spin labeling, and protein aggregation in detergent or liposomes.After its development, these protocols will allow obtaining distance and dynamic changes to explore mechanistic models for TRPV1 polymodal gating.

purification-and-reconstitution-of-trpv1-for-spectroscopic-analysis

Research

JoVE Journal

Biochemistry

7.9K Görüntüleme.  University of Tennessee Health Science Center. Here in the lab, we think that using a spectroscopic approach can help answering key questions in the transient receptor potential ion channel family. Such as determining the dynamic informational changes associated with polymodal activation. The main advantage of this protocol is that allow milligram of pure and functional remaining ion channel for electron paramagnetic resonance and double electron electron resonance epitroscopy.This method can provide insight into the conformational...