Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

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 donor vector²⁸ containing 8x histidine-maltose-binding protein (MBP)-tobacco etch virus (TEV) for expression and purification (Figure 2A). eTRPV1 single–cysteine mutants were generated using site-directed mutagenesis. Vector and channel sequences are specified in the Supplementary File 1: Supplementary Methods.

1. Design mutagenesis primers with online tools²⁹.
2. Mix in a 0.2-mL tube: 5 μL of 10x reaction buffer, 1 μL of dNTP mix (100 mM), 1 μL each of 10 μM forward and reverse oligonucleotides, 1 μL of 100 ng/μL eTRPV1 template DNA²⁶, 1.5 μL of DMSO, 1 μL of mutagenesis enzyme, and 38.5 μL of deionized water. Spin the mixture before placing the tubes in the thermocycler.
3. Perform PCR mutagenesis.
   1. Perform (a) denaturation at 95 °C for 2 min, (b) denaturation at 95 °C for 20 s, (c) annealing at 60 °C for 10 s, and (d) elongation at 68 °C for 4 min (30 s per 1 kb). Repeat steps b to d for 18 cycles, then perform elongation at 68 °C for 5 min, and maintain the reaction at 4 °C until further use.
4. Add 2 μL of DpnI enzyme to the reaction; mix and then incubate at 37 °C for 30 min.
5. Perform the transformation
   1. Thaw E. coli competent cells on ice.
   2. To a pre-cooled 14-mL sterile tube, add 75 μL of competent cells and 10 μL of DpnI-treated PCR mixture. Mix gently.
   3. Incubate reaction on ice for 30 min. Maintain the tube at 42 °C for 45 s and then immediately place it on ice for 2 min. Plate the mixture on Luria Broth (LB)-agar plate containing carbenicillin (0.2 mg/mL). Incubate the plate overnight at 37 °C.
   4. Harvest at least three single colonies from the plate and inoculate each in 4 mL of LB containing carbenicillin (0.2 mg/mL) using a 14-mL sterile tube. Incubate cultures overnight at 37 °C and shake at 250 rpm.
   5. Spin down overnight cultures at 8,000 x g for 10 min and extract DNA following the instructions of commercially available mini-preparation kits.
   6. Verify the presence of single-cysteine mutants by standard automated DNA sequencing (see Table of Materials).

2. Functional Analysis of eTRPV1 and Single-cysteine Mutants

1. HEK293 cell line transfection³⁰
   1. Culture HEK293 cells in 6 -well plates in 2 mL of high glucose medium (DMEM) per well (37 °C, 5% CO₂, 95% Humidity). Prepare HEK293 cells that are 70 ‒ 80% confluent.
   2. Prepare the transfection mixture in two separate 1.5-mL micro-centrifuge tubes.
      1. For reaction A, add 0.5 µg eTRPV1 or cysteine mutant-containing pMO to 250 µL of minimal essential medium (e.g. Opti-MEM). For reaction B, add 3 µL of transfection reagent to 250 µL of minimal essential medium. Incubate each reaction for 5 min at room temperature (RT).
      2. Mix reactions A and B with a 1-mL pipette. Incubate the mixture for 45 min at RT.
   3. Remove 500 µL of culture media from the 70 – 80% confluent well and add 500 µL of the reaction mixture. Store plates overnight for Ca²⁺ imaging (37 °C, 5 % CO₂, and 95% humidity).
2. Ca²⁺ imaging in HEK293 cells³⁰
   1. Clean 12-mm diameter glass coverslips with ethanol and water and place them into a 24-well plate. 
   2. Add 75 µL of poly-l-lysine in the center of each coverslip and store the plate for 45 min (37 °C, 5% CO₂, 95% humidity). 
   3. Remove the excess poly-l-lysine and wash coverslips three times with phosphate-buffered saline (PBS) to remove any leftover. 
   4. Once the coverslips are ready, proceed to detach cells from the 6-well plate.
   5. Remove the media and add 500 µL of warm PBS (37 °C) for washing.
   6. Remove PBS, add 500 µL of trypsin, and incubate for 1 min.
   7. Add 500 µL of warm culture media (37 °C) to stop the digestion reaction and mix with the pipette; avoid forming bubbles. If needed, adjust the cell concentration diluting this resuspension with more culture media.
   8. Seed 250 µL of transfected HEK293 cells (70% confluent) plus 250 µL of culture media (500 µL final volume per well) on the pre-treated coverslips and let them settle down (for attachment) for 1 – 2 h in the incubator (37 °C, 5 % CO2, 95% humidity). 
   9. In the meantime, prepare a loading solution by adding 0.02% pluronic acid and 1 µM Fluo-4-AM to a Ringer’s buffer. Mix the solution well.
   10. Remove the HEK293 culture media from the well and add 500 µL of the loading solution. Incubate cells for 1 h (37 °C, 5% CO₂, 95% humidity). Wash the Fluo-4-loaded cells twice with warm Ringer’s buffer.
   11. Place a coverslip with loaded cells into a 10-mm Petri dish in a microscope stage (using a 10X objective; 494 nm excitation and 506 nm emission) to perform intracellular Ca²⁺ measurements. 
   12. Measure changes in fluorescence intensity after perfusing respective ligands (e.g., 10 μM capsaicin) using commercially available software (see Table of Materials). 
3. mRNA and Xenopus laevis oocytes preparation³⁰
   1. Linearize eTRPV1 and single-cysteine mutants-containing pMO with PmeI for 1 h at 37 °C. Clean DNA following the instructions of commercially available purification kits.
   2. Use linearized and cleaned DNAs to synthesize capped RNAs with a commercially available kit compatible with T7 RNA polymerase.
   3. Clean capped RNAs according to commercially available purification kits. Quantify the RNA amount by measuring the absorbance at 260 nm wavelength.  
   4. Transfer 5 ‒ 10 mL of X. laevis oocytes (commercially available) into a 50-mL conical tube containing 20 mL of ND96 solution (96 mM NaCl, 2 mM KCl, 5 mM HEPES, 1 mM MgCl₂; pH 7.4) with 1 mg/mL collagenase and nutate for 50 min at RT.
   5. Rinse the oocytes in ND96 until the solution is clear; add fresh collagenase and shake for 30 min at RT. Examine the oocytes under a stereo microscope and stop the digestion when the outer follicular layer (glossy layer with red blood vessels) is absent in most oocytes (90%). Rinse oocytes in ND96 until the solution is clear.
   6. Transfer oocytes to a 50-mL polystyrene tube with ND96 plus 1 mM CaCl₂ and shake for 1 h. Under-digested oocytes will stick to the polystyrene tube.
   7. Wash oocytes with ND96 plus 1 mM CaCl₂ and supplement solution with 50 µg/mL of gentamicin and 50 µg/mL of tetracycline.
      Note: Protect tetracycline-containing solutions from light exposure.
   8. Select large oocytes without damaged membranes and displaying a clear separation between the animal and vegetable poles. Let oocytes recover for 4 h before microinjection at 16 °C.
   9. Use glass pipettes (O.D.: 1.11 mm, I.D.: 0.5, and length 8.8 cm) to inject 1-5 ng of mRNA from eTRPV1 and single-cysteine mutants into oocytes using a nanoliter-injector system (46 nL/s) and a stereo microscope (2X magnification). Store oocytes at 16 °C in ND96 supplemented with CaCl₂ and antibiotics (gentamycin/tetracycline 1x).
   10. After 72 h, measure macroscopic current using a two-electrode voltage-clamp (TEVC) acquisition system³¹.
   11. Pull borosilicate glass pipettes (0.3 MΩ) and fill them with 3 M KCl.
   12. Place the oocyte in the recording chamber using a transfer pipette and perfuse the bath solution (120 mM NaCl, 2 mM KCl, 2 mM MgCl₂, 1 mM EGTA, and 10 mM HEPES solution; pH 7.4) at low speed.
   13. Immerse both electrodes in the bath solution to adjust their pipette offsets. Gently push the pipette electrodes (borosilicate glass pipettes; O.D.: 1.5 mm, I.D.: 1.10, and length 10 cm) into the oocyte until a tiny indentation is observed under a stereo microscope (2X magnification), as well as a change in the membrane potential.
   14. Change the amplifier settings to recording mode and measure macroscopic currents while applying a voltage-clamp ramp from -80 to +80 mV for 1 s. Load the perfusion system with the solution used in step 2.3.12 at pH 7.4 (as a control) and at pH 5 to check eTRPV1 activity.

3. Generating the Recombinant Bacmid and Baculovirus for Protein Expression

1. Bacmid
   1. Transform 100 μL of DH10Bac E. coli competent cells with 7.5 ng of the recombinant donor vector containing eTRPV1 and/or single-cysteine mutants.
   2. Add 900 μL of SOC media (20 mg/mL Tryptone, 5 mg/mL yeast extract, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, and 20 mM glucose) and incubate for 6 – 7 h at 37 °C and 225 rpm.
   3. Seed 100 µL directly from the mixture, and from serial dilutions (1/10 and 1/100), in LB-agar plates containing 100 μg/mL X-gal, 40 μg/mL IPTG, 7 μg/mL gentamicin, 10 μg/mL tetracycline and 50 μg/mL kanamycin. Incubate the plates for at least 48 h at 37 °C.
   4. Select white colonies that are 2 mm 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.
   5. Harvest at least three single colonies and transfer them into a 14-mL sterile tube containing 4 mL of LB media supplemented with 7 μg/mL gentamicin, 10 μg/mL tetracycline, and 50 μg/mL kanamycin. Incubate cells overnight (~16 h) at 37 °C and 250 rpm.
   6. Take 1.5 mL of the overnight cultures and isolate recombinant bacmid DNA (refer to the Supplementary File 1 for detailed protocol). Store the bacmid at 4 °C for up to two months.
      Note: Prepare the glycerol stocks of DH10Bac E. coli containing the bacmid DNA. Take 300 μL of culture and add 200 μL of glycerol at 50% (final glycerol concentration of 20%). Store the aliquot at -80 °C until further use.
   7. Verify the presence of eTRPV1 in the recombinant bacmid using PCR (refer to Supplementary File 1 for detailed protocol).
2. Baculovirus
   Note: For this procedure, transfect Sf9 insect cells in a 6-well format. All amounts and volumes are given on a per-well basis. Avoid the use of antibiotics.
   1. Plate 1 x 10⁶ Sf9 cells in 2 mL of insect cell media. Allow cells to attach for at least 45 min.
   2. Dilute 1 μg of recombinant bacmid DNA in 100 μL of insect cell media. Mix gently.
   3. Dilute 6 μL of transfection reagent (TR) in 100 μL of insect cell media. Mix gently.
   4. Combine the DNA and TR solutions (from steps 3.2.2 and 3.2.3, respectively). Mix gently and incubate for 30 min at RT.
   5. Add 0.8 mL of insect cell media to the DNA/TR mixture; mix gently.
   6. Remove the insect cell media from the Sf9 cells and wash once with 1 mL of fresh medium.
   7. Remove the wash medium and add DNA/TR mixture (3.2.2 ‒ 3.2.5) to the cells. Incubate cells at 27 °C for 5 h (without agitation).
   8. Remove the transfection mixture and replace with 2 mL of insect cell media containing 0.5% fetal bovine serum (FBS). Incubate cells at 27 °C for five days.
      CAUTION: Prevent evaporation of the cell media by filling the empty wells with media and covering the 6-well plate with two layers of paraffin film.
   9. Transfer the cell culture media into a 15-mL centrifuge tube. Isolate the first passage (P1) of viral stock by spinning the tube at 8,000 x g for 15 min at 4 °C. Transfer the supernatant to a clean 15-mL centrifuge tube and immediately store it at 4 °C.
      Note: Protect the virus from light exposure by covering the tube with aluminum foil.
   10. For the second generation (P2) of viral stock, put a 25-mL suspension culture of insect cell media at 1 x 10⁶ Sf9 cells/mL containing 2% FBS into a 125-mL flask (plain bottom and vented). Add 25 μL of the P1 virus to the 25 mL culture.
   11. Incubate the culture at 27 °C for 72 h.
   12. To harvest the P2 viral stock, transfer the culture to a new 50-mL tube and centrifuge it at 8,000 x g for 15 min at 4 °C.
   13. Transfer the supernatant to a new 50-mL tube and immediately store it at 4 °C.
       Note: Protect the virus from light exposure by covering the tube with aluminum foil. Perform a small-scale experiment to titrate the P2 virus/Sf9 ratio for optimal TRPV1 expression (refer to Supplementary File 1 for a detailed protocol, Section 4 titled “Small-scale experiment for P2 virus.”)
   14. For large-scale protein expression, set a 1-L Sf9 cell suspension culture at 2 x 10⁶ cells/mL in insect cell media supplemented with 0.5% FBS into a 2.8-L borosilicate glass flask.
   15. Add 1 mL of P2 virus stock to the 1-L culture and incubate it at 27 °C for 72 h. 
       Note: On the third day, cell density should be at approximately 1.5 to 2.5 x 10⁶ cells/mL.

4. eTRPV1 and Single-cysteine Mutant Purification

1. Membranes isolation²⁸
   1. Harvest the 1-L insect cell suspension culture by spinning at 4,500 x g, 4 °C, for 20 min. Weigh the cell pellet (it is expected to be around 6 – 9 g).
   2. Resuspend the cell pellet in 25-mL of ice-cold Buffer A (36.5 mM sucrose, 2 mM tris(2-carboxyethyl)phosphine (TCEP), and 50 mM Tris; pH 7.4) in the presence of protease inhibitors (1 mM phenylmethyl sulfonyl fluoride (PMSF), 3 μg/mL leupeptin, 3 μg/mL aprotinin, and 1 μg/mL pepstatin). Disaggregate cell clumps to obtain a homogeneous suspension and rotate at 4 °C for 20 min.
   3. Break the cells using a manual (dounce tissue grinder) or a high-pressure homogenizer. Remove the cell debris by centrifugation at 8,000 x g for 20 min at 4 °C.
      Note: Keep the lysed cells and tubes on ice during the whole process.  
   4. Collect supernatant and centrifuge at 100,000 x g for 30 min at 4 °C. Discard supernatant and resuspend the membrane pellet in a total volume of 20 mL of ice-cold Buffer B (150 mM NaCl, 10% glycerol, 2 mM TCEP, 50 mM HEPES; pH 7.4), supplemented with protease inhibitors (as in step 4.1.2).
   5. Aliquot 20 mL of membrane suspension in 50-mL tubes and flash-freeze in liquid nitrogen. Store samples at -80 °C until further use.
2. Protein purification^26,28
   1. Thaw the membrane aliquots on ice and add 3 mL of n-Docecyl-β-D-Maltopyranoside (DDM) per tube (200 mM stock). Rotate the tube for 2 h at 4 °C.
   2. Centrifuge the sample at 100,000 x g for 30 min at 4 °C. Collect the supernatant and add 1 mL of clean wet amylose resin. Rotate the mixture for 2 h at 4 °C. Load the protein/amylose mixture in gravity flow chromatography columns.
      Caution: Do not allow the resin to dry during washes.
   3. Wash the protein-bound amylose resin with 10 times the bed volumes of ice-cold Buffer C (150 mM NaCl, 10% glycerol, 50 mM HEPES, 0.5 mM DDM, 0.1 μg/mL asolectin, 0.5 mM TCEP; pH 7.4). Wash with 10 bed volumes of ice-cold Buffer D (150 mM NaCl, 10% glycerol, 50 mM HEPES, 0.5 mM DDM, 0.1 μg/mL asolectin, pH 7.4).
      Note: Prior to washing, degas Buffer D without detergent or lipids, purging with nitrogen. After degasification, add DDM and asolectin.
   4. Elute eTRPV1 protein with 0.5 mL fractions, up to 5 mL, of ice-cold degassed Buffer D, supplemented with 20 mM maltose. If possible, perform the washes and elution at 4 °C.
   5. Quantify the protein amount by measuring the absorbance at 280 nm wavelength.
      Note: This protocol yields 0.5 to 1.0 mg of protein per liter of culture. Protein yield could vary for each eTRPV1 single-cysteine mutant. For EPR and DEER experiments, grow 4 ‒ 6 L of culture.

5. eTRPV1 Single-cysteine Mutant Site-Directed Spin Labeling

1. Concentrate the eTRPV1-containing fractions using a centrifugal filter unit (cutoff = 100 kDa) up to 2 ‒ 2.5 mg/mL for labeling (cycles of 7,000 x g for 2 min at 4 °C).
2. Add a 10-fold molar excess (3 times, every 30 min) of (1-oxyl-2,2,5,5-tetra-methylpyrrolidin-3-yl) methyl methanethiosulfonate (MTSSL) spin label from a 100-mM stock solution in DMSO to the concentrated protein (eTRPV1 monomer: MTSSL in 1:10 molar ratio)¹⁷. Keep the reaction in the dark at RT for 1 h 30 min, followed by overnight incubation at 4 °C.
   Note: In this step, MBP could be removed by adding TEV protease during the overnight spin-labeling incubation.
3. Load spin-labeled eTRPV1 on a size exclusion chromatography column equilibrated in Buffer E (150 mM NaCl, 20 mM HEPES, 0.5 mM DDM; pH 7.4), controlled by a fast protein liquid chromatography (FPLC) system. Collect fractions containing eTRPV1 tetramer.
   Note: Do not include 10% glycerol in this step, as it reduces the reconstitution efficiency.
4. Quantify the protein amount by measuring the absorbance at 280 nm wavelength.
5. Evaluate the purity of the sample by running a SDS-PAGE gel (stain-free gel, 4 ‒ 20%). The sample is now ready for spectroscopic measurements in solution and/or proteoliposomes.

6. eTRPV1 Spin-labeled Single Cysteine Mutant Reconstitution

1. Dry 10 mg of asolectin using a rotary evaporator under vacuum (≤100 mbar) for 1 h at 40 °C. To make asolectin liposomes, add 1 mL of Buffer F (200 mM NaCl, 5 mM MOPS; pH 7.4) and sonicate the mixture for 15 min or until the sample is homogeneous.
2. Destabilize the liposomes by adding DDM to a final concentration of 2 mM and incubate for 30 min at RT.
3. Concentrate the labeled protein at 2 mg/mL and add it to the liposomes using a 1:5 protein/Lipid ratio (mass:mass).
   CAUTION: It is important to keep the aforementioned protein concentrations, since spin-labeling efficiency decreases at low concentrations and at high the protein might precipitate.
4. Add Buffer F to adjust the protein/Liposome mixture to the DDM critical micelle concentration (CMC) and incubate overnight at 4 °C with gentle agitation.
5. Double the volume of the protein/Liposome mixture with Buffer F and remove detergent by sequentially adding three aliquots of nonpolar polystyrene adsorbent beads (30 mg, 50 mg, and 80 mg) at 1-h intervals with gentle agitation at RT.
6. Load the mixture onto a column filter (gravity flow chromatography column) to remove the beads and transfer the proteoliposomes mixture to an ultracentrifuge tube. Centrifuge the samples at 100,000 x g for 1 h at 4 °C. Discard supernatant and resuspend the pellet in 30 μL of Buffer F.
   Note: Samples are ready for spectroscopic analysis and injection into Xenopus oocytes. If the samples cannot be used the same day, flash-freeze them in liquid nitrogen and store at -80 °C.
7. Proteoliposomes-Xenopus oocyte electrophysiology^26,32
   1. Inject 50 nL of different dilutions of spin-labeled proteoliposomes into Xenopus oocytes as described in section 2.2.
   2. Perform TEVC measurements after 12 h of injection as described in section 2.3.10.
8. Proceed to perform spectroscopic measurements and analysis (section 7).

7. DEER and EPR Spectroscopies

1. Double Electron-Electron Resonance (DEER) Spectroscopy.
   1. Perform DEER measurements in a pulsed EPR spectrometer operating at Q-band frequency (34 GHz) and equipped with a 10-W amplifier with the dead-time free four-pulse sequence at 83° using manufacturer-supplied software³³.
   2. Supplement 50 µM of detergent-purified eTRPV1 spin-labeled cysteine mutants in Buffer E (step 5.5) with 30% (v/v) glycerol for cryo-protection.
   3. Load the sample into a sealed quartz capillary tube and spin to the bottom of the tube by brief low-speed centrifugation (100 x g).
   4. Place the capillary tube into liquid nitrogen to freeze the sample. The samples can be stored at this point at -80 °C for later measurement.
   5. Place the sample directly into the microwave resonator and let it re-equilibrate at -83° for 10 ‒ 20 min.
   6. Measure the sample using a standard four-pulse DEER protocol, (π/2)mw1–τ1–(π)mw1–τ1–(π)mw2–τ2–(π)mw1–τ2–echo³⁴. The pulse lengths for (π/2)mw1 and (π)mw1 are 10 and 20 ns, respectively, and 40 ns for (π)mw2. Set the frequency separation at 63 MHz.
      Note: Primary DEER decay data can be analyzed with a home-built software (e.g. in Matlab) that assumes a sum of Gaussian distributions to describe the distances between spin labels^19,35.
2. Continuous-wave (CW) EPR spectroscopy
   1. Perform CW EPR experiments at RT on an X-band (9.6 GHz) spectrometer using the manufacturer software.
   2. Start the instrument by turning on the water chiller, the console, and the magnet power supply. Connect the software to the instrument, place the instrument in tune and wait at least 30 min for the instrument to warm up.
   3. Load 20 µL of the sample from step 5.5 into a 25-µL glass capillary tube using capillary action and seal the end of the tube with sealant.
   4. Load the capillary tube into the microwave cavity and critically couple the resonator either manually or automatically using the auto-tune function of the software.
   5. Collect the first derivative spectra under standard instrument conditions: 100 kHz microwave modulation, 1.6 G magnetic field modulation, and 10 mW microwave power.
   6. For data analysis and presentation, correct the spectra against the background and normalize them by dividing the spectra by the peak-to-peak value of the double integral.
   7. Determine the spin-label mobility by measuring the inverse of the central line width of the first derivative absorption spectra (ΔH_o^-1)³⁶.