The overall goal of this procedure is to provide a detailed description of techniques for the functional and structural study of the effect of lipids on membrane proteins. This is accomplished by first expressing and purifying of voltage gated potassium channel. The second step is the ion channel reconstitution.
Next, the electrical recording from the ion channels in lipid bilayers is performed for the ion channel functional study. The final step is the crystallization of the ion channels in the membrane for structure determination. Ultimately, biochemical assays, electrophysiological method, and electron crystallographic study are used to investigate the lipid effects on the voltage sensor movement of ion channels.
Generally, people new to this method will struggle because of many technical details in protein reconstitution and bio recordings. Hopefully this film, the process will help you go through the whole procedure and succeed. The demonstration of the whole procedure will be performed by Dr.Hui Jim from my laboratory.
Prior to the lipid preparation was a 14 milliliter disposable glass test tube, a screw kept glass tube, and a 250 microliter glass syringe with chloroform. Then transfer 10 milliliters of chloroform into the test tube. To prepare POPE and POPG liposomes transfer 3.75 milligrams of POPE and 1.25 milligrams of POPG in chloroform into the screw capped glass tube.
Dry the lipids under a continuous stream of argon gas when no visible chloroform is left. Try the lipid further under room vacuum for one hour. Next, add 440 microliters of low salt buffer or water into the dried lipid vortex.
The tube to hydrate the lipids. The lipid suspension should look whitish and turid. After that, sonicate the lipid suspension in an ice cold bath sonicate until the vesicle solution becomes translucent.
Then at 50 microliters of three molar potassium chloride and 10 microliters of 0.5 molar DM to the mixture so that the final lipid suspension has 300 millimolar potassium chloride and 10 millimolar DM incubate the solution in a rotator for two hours at room temperature to allow the formation of lipid detergent mixed M cells. After the incubation, the suspension should become a little turid due to the detergent induced fusion of the small ELLA vesicles. When the protein is concentrated to more than two milligrams per milliliter, add the listed components, the lipid detergent mixture to make one milliliter of final volume.
Then incubate the protein lipid detergent mixture in the glass tube in a rotator for another two hours. The preparation of the KVAP in mixture with the detergent solubilized DTaP or DOGS, is similar to the lipid preparation for P-O-P-E-P-O-P-G vesicle. However, the sonication of hydrated lipids into small unli vesicles takes a longer time than that for the P-O-P-E-P-O-P-G lipids, the DOGS vesicles may slowly fuse with each other and form small oily droplets to solubilize the dote or DOGS completely.
The sonicated vesicles are mixed with 10 millimolar DM and 40 millimolar beta og. The lipid detergent mixture is incubated overnight at room temperature and the mixture should be fairly clear and not have any small particles or droplets. Next, mix the KVAP protein with the detergent solubilized DTaP or DOGS in a protein to lipid ratio that is less than or equal to one to 10.
The protein detergent lipid mixture is allowed to incubate a room temperature for two to three hours with continuously orbital rotation. To demonstrate the slow removal of the detergents that have relatively high CMC, such as DM and Beta og. First prepare two liter dialysis buffer for each one milliliter mixture.
Then cut out a suitable length of dialysis tubing and wash it with DI water to check and make sure that there is no leak in the tubing. Next, load the protein lipid detergent mixture into the dialysis tubing. Clamp both ends of the tubing with minimal space left above the solution.
Place the loaded tubing on a stirring plate in the dialysis buffer so that the tubing rotates slowly in solution. Change the dialysis buffer once every eight hours. Now prepare the polystyrene beads for the removal of detergents that have low CMC by first wing 0.5 grams of dry beads.
Then put them into a 50 milliliter Corning tube and add 20 milliliters of methanol sonicate the solution in a bath Sonicate for five minutes to remove trapped bubbles and spend on the beads at 5, 000 RP for five minutes afterward, decant the methanol completely and repeat the wash with ethanol and milli Q water. Then store the washed beads in 20%ethanol at four degrees Celsius and change it to a detergent free buffer before use. Estimate the amount of detergents in the protein lipid detergent mixture to be treated and calculate the amount of beads needed to remove the detergents.
Weigh the wet beads without excess water and add them directly to the protein mixture and wait for at least 15 to 20 minutes for the beads to be fully effective. To remove 8.7 milligrams of DDM in one milliliter of solution, a total of 87 milligrams of polystyrene beads such as bio beads, SM two is divided into five equal portions. Mix the protein lipid detergent mixture with each fraction for 20 to 30 minutes with constant end over end rotation at room temperature.
Spin down the beads, then transfer the supernatant to the next fraction of beads and repeat till the end. In this step, begin by cleaning a glass test tube and amber vial with a Teflon surfaced screw cap and a set of glass syringes with chloroform. Then dry the amber vial under a stream of argon gas.
Next transfer, 0.75 milligrams of POPE and 0.25 milligrams of POPG in chloroform into the ember vial and evaporate the chloroform with argon gas. After that, add 0.2 milliliters of pentane to the vial to dissolve the dried lipids and dry completely to remove residual chloroform. Next, add 50 microliters of decane to the vial to dissolve the dried lipid.
Now, paint the round hole at the cylindrical surface of the bilayer cup with the lipids without getting any lipid solution into the hole. Wait for one to two minutes for the decade to evaporate completely to perform electrical recordings from KVAP channels in lipid by layers. Insert the cup into the recording chamber, put in the salt bridges and connect the electrodes to the two sides of the recording cup.
Add the cyst solution to the electrode holes and the CYS and trans solution to the outside and inside cups in the chamber. Then adjust the potential offset between two sides of the cup to less than two millivolts, and then paint lipid across the hole in the cap. Wait till the membrane thins out and becomes relatively stable.
As soon as the membrane becomes stable. Shoot 0.5 to one microliter of channel containing vesicles by positioning the fine end of a P two pipette tip right above the hole. The vesicles fall down across the hole to the bottom of the cup.
To test the KVAP channels in the bilayer. A short pulse of 80 millivolts is delivered from the holding potential of minus 80 millivolts due to the fast inactivation and slow recovery from an activation a long interval, about 120 seconds for channels in PEPG membranes is given between two pulses. Once the current looks good in size, balance the ion concentrations in the solutions on both sides of the membrane by adding additional ion salts to the low concentration side.
Here is an image of a negatively stained 2D crystal, the middle of crystal optimization. The crystals were stained with 6%ammonium moate, pH 6.4 plus 0.5%triose due to the sugar they sometimes piled up with each other. The black square box here designates an area that gives rise to the diffraction pattern with diffraction spots.
Going to about 20 angstrom here is a cryo EM image of a 2D crystal from a sample similar to the one shown before. The specimen was mixed with 3%TLOs and frozen by direct plunging and imaged under a cryo EM at 50, 000 decks. It is clear that there were local defects in the crystal packing.
Here is another cryo-EM image of a further optimized crystal. The specimen was embedded in 0.75%tannin and 10%tree hellos, and the image was taken at 50, 000 x. The straight lines in the tight packing suggested that the channels were well ordered in this type of crystals, whereas the two black arrows mark the square vectors.
After watching this video, I hope that you will get a good sense about how to handle lipid and protein properly, and this video hopefully will help you to study lipid protein to action in membrane more efficiently.
