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

The overall goal of this protocol is to demonstrate the purification, site directed spin labeling, and reconstitution of a penameric ligand-gated channel for electron paramagnetic resonance studies. Studying membrane protein dynamics in a physiologically relevant environment is crucial to fully understand how this membrane transport protein works. And because of these needs, I think site directed spin labeling and EPR technique is ideally suited.

The main advantage of this technique is that it's not limited by the size of the protein. Therefore, site directed spin labeling and EPR spectroscopy would be a technique of choice if you're wanting to study large protein dynamics or proteins reconstituted in membranes. Introduce single cysteine mutations in the GLIC gene by site directed mutagenesis.

Set up an overnight culture of the GLIC cysteine mutant as described in the text protocol. Next, add 100 milliliters of sterile potassium phosphate buffer to the autoclave terrific broth media. Then, add sterile glucose, Kanamycin, and 10 milliliters of the overnight culture.

Incubate at 37 degrees Celsius in a shaker at 250 rpm. Add 50 milliliters of glycerol and continue shaking the culture until the optical density at 600 nanometers reaches 1.0 to 1.2. After inducing the cells with 200 micromolar IPTG, reset the shaker back to 250 rpm and incubate further for approximately 16 hours at 18 degrees Celsius.

Harvest the cells in one liter centrifuge bottles by spinning at 8500 times G, four degrees Celsius, for 15 minutes. Decant the supernatant and weigh the bottle with the cell pellet as described in the text protocol. Resuspend the bacterial pellet in 150 milliliters of ice-cold buffer A per one liter of culture.

Homogenize the cells by passing them through a cell disrupter in a four degrees Celsius cold room. Repeat the process three times so that most of the bacteria is lysed. Centrifuge the cells at 14, 000 times G for 15 minutes.

Then, pipette the supernatant out and transfer into a clean centrifuge tube. After centrifuging the supernatant at 168, 000 times G for one hour, carefully decant the supernatant without disturbing the membrane pellet. Pull the membrane pellet from one liter of culture and resuspend it in Buffer A, supplemented with 10%glycerol, to a final volume of 50 milliliters.

Add 0.5 grams of DDM to the membrane suspension and nutate for two hours at four degrees Celsius. After membrane solubilization, remove cell debris from the lysate by centrifugation at 168, 000 times G for one hour. In the meantime, prepare amylose resin.

Transfer three milliliters of resin using a pipette into an empty polypropylene chromatography column. Wash the resin by passing 10 bed volumes of water three times, and then 10 bed volumes of Buffer A, containing 0.5 millimolar DDM. After centrifugation, gently take the supernatant out using a pipette and transfer to a clean 50 milliliter conical tube.

For batchwise binding, add pre-equilibrated amylose resin to the conical tube. Bind the extracted protein to amylose resin by nutating the mixture for two hours at four degrees Celsius. Next, pass the entire slurry through a chromatography column and collect the flow-through.

Dilute the GLIC protein with 10 milliliters of Buffer A containing 40 milliomolar maltose in addition to 0.5 millimolar DDM and 05 milliomolar TCEP. TCEP prevents oxidation of cysteine side chains. Collect the entire eluite.

Concentrate the eluted protein using a centrifugal contentrator to between four and six milligrams per milliliter. Add HRV 3C protease and incubate overnight at four degrees Celsius. To perform site directed spin labeling, first add tenfold molar excess of MTSL spin label to the protease digested sample to result in a GLIC monomer to MTSL molar ratio of 1:10.

Following one hour incubation, add more spin label at a fivefold molar excess ratio and incubate for another hour. Pass the sample through a size exclusion fast protein liquid chromotography, or FPLC column, that is pre-equilibrated with Buffer A and 0.5 millimolar DDM to separate the cleaved MBP tag and the excess free spin label from GLIC pentamers. Concentrate the protein solution using a centrifugal concentrator to a final concentration of approximately eight to ten milligrams per milliliter and place it on ice.

The sample is now ready for reconstitution. To prepare the liposomes, rinse a clean 25 milliliter round-bottom flask with five milliliters of chloroform and dry the flask in a stream of nitrogen gas in the fume hood. Then, transfer 10 milligrams of asolectin into the flask and dry the lipids under a continuous stream of nitrogen gas.

When the chloroform has evaporated, place the flask in a vacuum for one hour to ensure complete drying. Then, add one milliliter of reconstitution Buffer A to the dried lipid and vortex the flask vigorously to get the lipid pellet into suspension. To prepare small unilamellar vesicles, sonicate the lipid suspension in a cold bath sonicator until the vesicle solution becomes more or less translucent and no clumps are observed.

To this mixture, add DDM to a final concentration of four millimolar and incubate at room temperature for 30 minutes. To reconstitute the protein, add the purified protein to the lipid mixture. Following incubation of the sample at four degrees Celsius for one hour with gentle rotation, dilute the sample to 15 milliliters with Buffer A.Remove the residual detergent in the suspension using polystyrene beads with hydrophobic pores that trap detergent.

Add 80 milligrams of clean beads and incubate the liposome suspension overnight on a nutator at four degrees Celsius. The next day, transfer the liposome to a 25 milliliter ultracentrifuge tube using a column filter to remove the beads and further dilute the sample to 25 milliliters. After centrifuging the samples at 168, 000 times G for two hours, decant the supernatant and resuspend the pellet using 100 microliters of Buffer B.To perform continuous wave EPR spectroscopy, first transfer the liposome suspension into a 200 microliter tube and pellet the samples using a centrifuge.

After discarding the supernatant, the sample is ready for EPR measurements. Remove as much buffer from the liposome sample as possible. To carry out buffer exchange, transfer 20 microliters of liposome pellet to a microfuge tube using a pipette.

Add 180 microliters of Buffer D and incubate at 42 degrees Celsius for five minutes. Centrifuge the sample, remove the supernatant using a pipette, and repeat the process three times to ensure complete buffer exchange. Gas permeable plastic capillaries are suitable for measurements of both spectral line shapes and solvent accessibility.

Tap the capillary onto the pelleted proteoliposome to draw the sample inside and seal the end with bone wax. Shown here is the biochemical characterization of spin labeled GLIC mutants through the gel filtration chromatogram of spin labeled GLIC after proteolytic cleavage of the MBP tag. The peaks correspond to the GLIC pentamer and free MBP.

The SDS page gel shows the uncut monomeric GLIC-MBP fusion protein before gel filtration, as well as the MBP and GLIC fractions pulled from gel filtration. A FRET-based assay for monitoring the aggregation state of GLIC reconstituted in various membrane lipids was performed. FRET measurements were done for samples after overnight reconstitution and after multiple cycles of freezing-thawing the samples.

In an inside-out patch excised from reconstituted asolectin vesicles, GLIC is activated by pH jumps using a rapid solution exchanger. The channels are seen to activate in approximately 10 milliseconds and desensitize with a time constant of one to three seconds. Structural rearrangements are observed during channel activation.

Here, continuous wave electron paramagnetic resonance spectra for a position in the pore-lining M2 helix displace changes in amplitude and blind shapes in response to pH changes. EPR spectroscopy is an unparalleled structure approach to measure conformational changes of membrane proteins in our native environment, and it provides a glimpse of molecular details of protein dynamics that are obscured from high-resolution X-ray structures or cryo-EM structures. While working with this technique, it is important to bear in our mind that mutational quite often lead to low and poor oligomeric stability of protein.

Therefore, you may need to come up with strategy to combat with these issues. Combining these findings from these EPR studies with the functional measurements can give us a more complete picture to study the structure-function relationship, and future technological advancements are aimed toward using this powerful approach to study more complicated human channels in more sophisticated lipid conditions.