Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

The overall goal of these three complementary experimental techniques, namely, yeast two-hybrid, Co-immunoprecipitation and chemical cross linking, is to assess protein-protein interactions and in particular, self-association, and to determine the stoichiometric composition of protein homo-oligomers. These methods can help answer key questions in the field of cardiopathies such as ultra-dry receptor oligomerization, underlying cardiac arrhythmias and sudden death. The main advantages of these techniques are their relatively high throughput screening, ease of use and highly reproducible results, while requiring minimal equipment and reagents.

The implications of these techniques extend toward therapy of arrhythmogenic cardiac disease, because they help establish the molecular mechanism of action of anti-arrhythmic drugs. Generally, individuals new to Co-immunoprecipitation will struggle because of potentially discarding the protein agarose beads, which are very small, when aspirating the supernatant during the centrifugation steps. In this yeast two-hybrid assay, yeast cells in mid-log phase are used for transformation.

Harvest the cells by centrifugation, resuspend each pellet in five milliliters of sterile water, and pool the cell suspensions. Centrifuge the cell suspension. After discarding the supernatant, resuspend the yeast pellet in one milliliter of freshly prepared, sterile one-x lithium acetate and TE.The competent yeast cells must be used within one hour of preparation.

Prepare plasmid samples by mixing bait and target plasma DNA with 100 micrograms of herring testes carrier DNA. To each 1.5 milliliter tube, add 100 microliters of the competent yeast suspension and 600 microliters of one-X lithium acetate and PEG solution, and vortex for about 30 seconds. Incubate at 30 degrees Celsius for 30 minutes, with shaking.

Add 80 microliters of dimethyl sulfoxide and mix well by gentle inversion. Heat shock for 15 minutes in a 42 degrees Celsius water bath, while mixing every two to three minutes. Chill the cell suspension on ice for two minutes, and then centrifuge to recover the yeast.

Resuspend each cell pellet in 100 microliters of one-X TE.Plate the cells on appropriate minimal SD medium plates to keep selective pressure on both bait and target plasmids. Incubate the plates upside-down at 30 degrees Celsius for four to five days. The yeast colonies are ready for this assay when they are about two millimeters in diameter.

Pipette 2.5 milliliters of freshly prepared Z buffer and X-gal solution to a clean 100-millimeter plate and place a cellulose filter paper in the plate. Place a new filter paper over the surface of the plate with the yeast colonies to be assayed. Use forceps to gently rub the filter paper onto the plate, and leave for approximately five minutes for the colonies to attach.

While using appropriate personal protective equipment, carefully lift the filter paper and submerge it into a pool of liquid nitrogen for 30 seconds. Let the frozen filter paper thaw at room temperature for two minutes. Place the filter paper with the colony side up on top of the pre-soaked filter paper inside the 100-millimeter plate and incubate at 30 degrees Celsius until blue colonies appear.

One day before transfection, feed HEK293 cells in a 100-millimeter petri dish, in order for cells to be 60 to 70 percent confluent the following day. Culture for 16 to 18 hours in a humidified incubator. On the day of transfection, dilute 24 micrograms of plasma DNA with 60 microliters of 2.5 molar calcium chloride and sterile de-ionized water to obtain a total volume of 600 microliters.

Vortex to mix. Add the plasma DNA plus calcium solution dropwise into a 50-milliliter tube containing 600 microliters of 2X HEPES-buffered saline while constantly and vigorously mixing by vortexing. Incubate for 20 minutes at room temperature to allow the formation of calcium phosphate plasma DNA complexes.

After the 20 minute incubation, vortex briefly, and add the solution dropwise onto the HEK293 cells to cover the whole surface area of the petri dish. Incubate the cells at 37 degrees Celsius in five percent carbon dioxide. After approximately six hours, change the growth medium and place the cells back in the incubator.

24 hours after transfection, harvest the cells as described in the protocol text. Resuspend the cell pellet in 500 microliters of ice-cold homogenization buffer and transfer the cell suspension into a 1.5 milliliter tube containing glass beads. Use a fine needle attached to a one-millimeter syringe to homogenize the cells on ice.

With the tube cap closed, pierce the cap with the needle and disperse the cell suspension vigorously through the glass beads. Centrifuge at 1500 times G for 10 minutes at four degrees Celsius to remove nuclei and unbroken cells. Save the supernatant representing the post-nuclear fraction for use in co-immunoprecipitation or cross linking experiments.

To being this procedure, solubilize the post-nuclear subcellular fraction with 0.5 percent chaps and incubate for at least two hours at four degrees Celsius with constant mixing. Centrifuge at 20, 000 times G for 10 minutes at four degrees Celsius to pellet the insoluble material. Save the supernatant.

This is termed the cell lysate. Prepare protein-A agarose beads in immunoprecipitation buffer as described in the protocol text. Add one microgram of the immunoprecipitating antibody to one protein-A containing tube.

As a negative control, add one microgram of non-immune IGG to the other protein-A containing tube. Incubate for at least two hours at four degrees Celsius, with constant mixing. Recover the beads by centrifugation and carefully discard the supernatant by pipetting.

Transfer 200 microliters of the cell lysate into each of the two tubes containing antibody and protein-A beads. Incubate for at least two hours at four degrees Celsius with constant mixing to allow for antigen-antibody binding. Recover the beads, and wash with 200 microliters of immunoprecipitation buffer.

Incubate for 10 minutes at four degrees Celsius with mixing. After three washes, carefully discard the supernatant by pipetting. Add 30 micorliters of protein loading buffer to the beads and centrifuge at 1500 times G for two minutes at four degrees Celsius.

Save the supernatant containing the alluded co-IP sample for subsequent SDS page and immunoblotting. To perform chemical cross-linking, centrifuge the post-nuclear subcellular fraction to pellet protein aggregates. Save the supernatant and divide into eight aliquots of 20 microliters each.

Add 0.0025 percent volume-per-volume glutaraldehyde to only one sample. Start the timer and let the glutaraldehyde react at room temperature for the indicated length of time. Stop the glutaraldehyde reaction at the specified time with two percent volume-per-volume hydrazine and add five microliters of 5X protein loading buffer to denature the proteins.

Yeast two-hybrid was used to screen overlapping constructs spanning the entire length of the ryanodine receptor peptide sequence for interaction with AD4L and end terminal fragment. Colony-lift filter paper beta-gal assays produced vivid blue-colored colonies only for the BT4L/AD4L pair, showing that AD4L interacts with itself. Pale blue colonies were detected for the BT8/AD4L pair, suggesting a secondary, weaker association with the extreme C-terminal domain.

Quantitative results from liquid beta-gal assays indicated robust BT4L/AD4L interaction, equivalent in strength to the known association between the P53 protein, pVA3 and the SV40 large T antigen, pTD1. The BT8/AD4L interaction was considerably weaker. The yeast two-hybrid findings are reinforced by co-immunoprecipitation experiments following transient expression of HA-tagged AD4L and cMyc-tagged BT4L in a mammalian cell line.

Direct immunoprecipitation of HA-tagged AD4L by an antibody 2 HA was observed, and cMyc-tagged BT4L was recovered only in the anti-HA immunoprecipitation. Chemical cross-linking was performed on cell homogenate expressing cMyc BT4L followed by SDS page and immunoblotting, using an antibody to cMyc. In addition to the 100 kilodalton monomer, a high-molecular mass protein band of approximately 400 kilodaltons was detected in a time-dependent manner, indicating ryanodine receptor and terminus tetramer formation.

While attempting mammalian cell transfection by calcium phospate precipitation, it's important to remember to constantly and vigorously vortex the phosphate buffer in a large tube for efficient aeration, while very slowly adding the plasma DNA consume solution. Following these procedures, other methods like confocal in mycroscopy in calcium imaging can be performed. These techniques should help answer additional questions, such as the localization and calcium release properties of ryanodine receptor channels in living cells.

After their development, these techniques paved the way for researchers in diverse fields of cell signaling to assess protein homo-oligomerization, a fundamental biological process that regulates the activity of transcription factors, enzymes, ion channels and receptors.