Signal transduction is one of the most important topics in biological and pharmaceutical research. It requires membrane proteins to transmit signals to their intracellular signaling partner protein. This protocol is designed to perform a systematic detergent screening in preparation of a signaling complex aiming for structure determination.
This protocol uses commonly available techniques in a well established biology laboratory and be easily performed by the beginners in the field. We comprised these methods to find the most critical parameter in preparation of membrane protein complex. To begin, thaw 30 grams of HEK293 GNT I-deficient cell pellet to room temperature and transfer it to a beaker.
Add 120 milliliters of PBS buffer containing protease inhibitor cocktail and homogenous using a Dounce homogenizer or an electric homogenizer at 13, 000 RPM for 30 seconds. Adjust the volume to 150 milliliters with PBS buffer. Gently add 10%DDM to the homogenized cells to give a final concentration of 1.25%Stir on ice for one hour.
After solubilization, centrifuge the cell lysate at four degrees Celsius and 150, 000 times G for 45 minutes to remove the unsolubilized debris. Transfer the supernatant to a 500 milliliter bottle and add 10 milliliters of the 50%1D4 immuno-affinity agarose resin. Gently mix the solubilized cell lysate and resin for four hours or overnight at four degrees Celsius to allow protein binding.
Load the lysate resin mixture to a open column to collect the resin. Wash the resin with 10 column volumes of wash buffer A to remove the protein contaminant. Close the valve and resuspend the resin with two column volumes of buffer A.Next, under dim red light condition, add 9-cis Retinal to the resuspended resin to a final concentration of 50 micromolar.
Gently mix at four degrees Celsius for four to 16 hours in the dark for Retinal binding. After that, open the value and remove the buffer from the column. Wash resin with 20 column volumes of buffer A, followed by 15 column volumes of buffer B to remove unbound Retinal.
Resuspend the resin in two column volumes of buffer B and then divide the resin suspension equally to 10 10 milliliter disposal columns. Remove the buffer from the 10 columns and then resuspend the resin each in one milliliter of buffer C for detergent change. Incubate for one hour on ice.
Repeat the washing and the incubation in buffer C one more time. Remove the buffer from the columns and then resuspend the resin in 0.8 milliliters of elution buffer for each column. Gently mix for two hours.
Collect the elution from each column into a tube. Resuspend the resin in 0.7 milliliters of elution buffer for each column. Gently mix for one hour.
Collect the elution from each column into the same tubes. In the dark, load the eluded protein to the quartz cuvette. Measure the spectrum of the protein sample.
Illuminate the protein directly in the cuvette for two minutes with light pass through of 495 nanometer long pass filter. Measure the spectrum of the illuminated sample. Perform the same measurement for all the protein samples purified in the other nine detergents.
Both dark and illuminated states. Under normal light, for each detergent condition prepare 100 microliters of rhodopsin at 0.7 milligrams per milliliter. Under dim red light, prepare 100 microliters of mixture of rhodopsin at 0.7 milligrams per milliliter and mini-GO at 0.2 milligrams per milliliter.
Supplement the mixture with one millimolar magnesium chloride. Illuminate the mixture with light from a 495 nanometer long pass filter and incubate for 30 minutes. Transfer the samples to the auto-sampler vials and place them in the sample tray.
Program a method file to automate sequential SEC runs for each sample with the auto-sampler loading 77 microliters of the sample to the column and the purifier eluding 25 milliliters of SEC buffer per run. Record the absorbance at 280 nanometers and 380 nanometers. Collect the peak fractions of rhodopsin and rhodopsin-mini-GO complex at the retention volume around 12.9 milliliters.
Analyze the left rhodopsin samples and the peak fractions of rhodopsin-mini-GO complex on four to 12%SDS denaturing gradient gels with Coomassie blue staining. Prepare a 200 microliter mixture of rhodopsin at one milligram per milliliter and PNGase F at 0.01 milligrams per milliliter. Mix well and incubate on ice overnight.
Prepare a 200 microliter mixture of rhodopsin at one milligrams per milliliter and Endo F1-13 at 0.01 milligrams per milliliter. Mix well and incubate on ice overnight. Analyze the digestion result by SDS-Page and Coomassie blue staining.
In this study, small scale purification setup yielded sufficient protein for further analyses. Ultraviolet visible spectra of rhodopsin show the dark state 9-cis Retinal bound rhodopsin in blue curves. After illumination, 9-cis Retinal is deproteinated and isomerizes into all trans-Retinal.
The ratios of absorbents at 280 nanometers over absorbents 488 nanometers and absorbents at 280 nanometers over absorbents at 380 nanometers depict the stability of rhodopsin purified in each detergent. Size exclusion chromatography profiles of rhodopsin and rhodopsin-mini-GO complex purified in 10 different detergents are shown here. The profile of the standard marker proteins is shown as overlay together with the DDM sample.
The interpretation of the peak profiles is shown for DMNG with the ideal scenario seen for DDM, DM, Cymal-6, Cymal-5, and OGNG. The magnified profile of the OGNG sample shows both rhodopsin and rhodopsin-mini-GO complex eluded around the same retention volume. The SDS-Page analysis of rhodopsin and SEC purified samples of rhodopsin-mini-GO complex is shown here.
This figure shows the SDS-Page analysis of rhodopsin deglycosylation with Endo F1 and PNGase F enzymes. It is critical to perform detergent screening in a systematic way, so that the impact of each individual detergent can be clearly compared without ambiguity. For protein of various quantity, thermal shift assay can also be used to study the impact of detergent on protein stability.
Thanks to this method, we are able to prepare stable protein complex and eventually solve the crystal structure lost in geo-assembly. It provided important insights into GPCR-G protein interaction specificity.
