The overall goal of the following experiment is to introduce a single informative probe into target G-Protein coupled receptors to facilitate structural and dynamic studies of signaling complexes. This is achieved by transecting cells with the plasmids necessary for expressing the required translational machinery to incorporate the unnatural amino acid zeto, phenylalanine, or zeto fee into an expressed GPCR. The GPCR zeto fee variance can then be applied in three different ways to investigate the structure and function of the receptor.
First, the zeto fee is used as a photo activate crosslinker in the GPCR, which enables the identification of the binding site of a tritiated ligand. Next, the zeto fee can be used as a reactive chemical handle in order to attach a peptide conjugated or fluorescent probe onto the GPCR via bio orthogonal labeling chemistries results are obtained that show use of the molecular probe zeto fee in targeted photo cross-linking, targeted epitope tagging and fluorescent labeling of GPCRs based on live cell and biochemical methods. The main advantage of this technique over existing methods like the use of photo affinity ligands or cysteine labeling, is the flexibility to site specifically introduce a biologically inert and reactive unnatural amino acid P zeto phenol alanine at a known position in a target receptor.
This method can help answer key questions in the GPCR signaling field, including mapping the sites of interaction in a receptor LIG complex in the native membrane environment, and facilitating single molecule fluorescent studies by identifying positions in A-G-P-C-R amenable to bio orthogonal labeling. Third, this method Can provide insights into the molecular mechanism of G protein car receptors and it can also be applied to other systems such as a variety of cellular and membrane proteins. Prior to starting this experiment, maintain HEC 2 9 3 T cells in KO's modified eagle medium, supplemented with 10%fetal bovine serum at 37 degrees Celsius in a 5%CO2 atmosphere.
Transfect cells at 60 to 80%confluence in a 10 centimeter plate with G pcr, r CD, NA using lipectomy plus reagent as described in the text protocol. The next day, replace the growth media with DMEM containing 10%FBS and 0.5 millimolar fee. 48 hours post transfection analyze expression as described in the text.
Proceed to photo, cross linking or labeling procedures as described in the remainder of this video. 48 hours post transfection aspirate the media from the cells and replace it with four milliliters of one x phosphate buffered saline or PBS return to 37 degrees Celsius for 15 minutes so that the cells detach from the plate. Then resuspend the cells with one XPBS and transfer to a Falcon tube centrifuge at 1500 RPM for two minutes.
To pellet the cells aspirate the supernatant before Resus suspending the cell pellet in one x Hanks buffered salt solution containing the tritiated ligand at saturating binding concentration prepared as described in the text protocol. Incubate the pellet at 37 degrees Celsius for the required length of time to ensure receptor ligand complex formation, which is dependent on the specific receptor and ligand. Next, transfer the cell suspension to a 24 well or 96 well plate.
Then photo activate the zeto fee using an ultraviolet, a lamp at 365 nanometers for 15 minutes at four degrees Celsius, maintaining the plate on ice to avoid sample heating and cell lysis. Following photo activation, transfer the cells to an einor tube and centrifuge to pellet the cells after centrifugation. Remove the supernatant and proceed to analysis.
Resuspend the cell pellet in lysis buffer, and incubate the cells for one hour at four degrees Celsius. Then centrifuge the cell lysate for 10 minutes at 16, 000 times G.Before transferring the supra natin to a new tube, add 1D four monoclonal antibody modified SROs two B resin to the supernatant and incubate overnight at four degrees Celsius to immuno purify the GPCR using the engineered C terminus 1D four monoclonal antibody epitope the next day, wash the beads several times with lysis buffer. Elute the samples with 1%SDS at 37 degrees Celsius for one hour with shaking.
Transfer a portion of the EENT to a 15 milliliter vial containing scintillation fluid and count on a beta scintillation counter to quantify the amount of specific binding of the tritiated ligand to the receptor. Resolve the remaining purified EENT by standard gel electrophoresis using a four to 12%SDS gel with standard protein ladder. Then semi-dry transfer to A-P-V-D-F membrane for immuno blotting.
Block the PVDF membrane with 5%milk in TBS buffer containing 0.05%Tween 20. Probe the membrane to confirm full length receptor expression with 1D four monoclonal antibody, followed by HRP conjugated anti-US IgG secondary antibody. Then treat the membrane with enhanced chemiluminescence reagent.
Expose the membrane to auto radiography film to visualize the bands. Next, cut the membrane with a razor blade to separate each sample lane and then cut at specific molecular weight markers. Transfer the membrane segments to vials containing scintillation fluid and then quantify the amount of tritium in each membrane segment by counting on a beta scintillation counter.
Identify the positive photo cross link with the detection of tritium at the apparent molecular mass equal to the sum of the GPCR and the ligand. After washing and counting cells pretreat A 96 well high binding clear bottom plate with 100 microliters of 10 micrograms per milliliter. Poly D lycine per well incubate for 30 minutes at room temperature.
Then wash repeatedly with one XPBS before allowing to air dry plate the transfected cells in 200 microliters at a density of 60 to 80, 000 cells per well. In the 96 well plate and return to 37 degrees Celsius in a 5%CO2 atmosphere. The next day.
Prepare the cells for labeling. Gently wash the cells three times with 100 microliters per well using one XPBS to remove any zeto fee containing growth media. Prepare a buffer containing 50 to 200 micromolar flag Tri AAL PHOSPHINE labeling reagent as described in the text protocol.
Apply 60 microliters to each well. Maintaining a set of wells without label treatment and buffer. Also include a wild type GPCR control to compare with a zeto feed GPCR variance.
Return the plate to 37 degrees Celsius between 30 minutes and four hours following incubation. Wash the cells three times with 100 microliters per well of blocking buffer to remove unreactive label completely. Next, apply 100 microliters per well of 4%paraform aldehyde to the cells after incubating for 20 minutes at room temperature.
Wash the cells three times with blocking buffer. Perform a standard cell surface EISA protocol to detect for labeled receptor and receptor expression by first incubating with primary antibody for 1.5 hours on ice. Follow this by a secondary antibody incubation at room temperature for one hour.
Next, add 100 microliters of diluted Anti-Flag M two antibody to detect the flag peptide epitope of the flag. Tri AAL phosphine labeling reagent. Also probe non label treated wells as a negative control.
Then add diluted anti CCR five 2D seven antibody to separate sets of wells to determine wild type or zeto fee. GPCR cell surface expression. Wash the cells three times with blocking buffer and incubate with 100 microliters of diluted HRP conjugated anti-US IgG antibody.
After carefully washing the cells five times with blocking buffer, add 50 microliters of detection buffer and incubate for 15 minutes at room temperature. Finally, collect spectral data on a fluorescence multi-well plate reader at an excitation wavelength of 530 nanometers and an emission wavelength of 590 nanometers. Begin the labeling procedure by lysing 10 million harvested cells expressing wild type or zeto fee.
Rod opsin variance in one milliliter of solubilization buffer for one hour at four degrees Celsius. Collect the sate fraction following centrifugation of the cell lysate at 15, 000 times G for 10 minutes. Then add 100 microliters of 1D four monoclonal antibody modified spheros two B resin to capture the receptor using the engineered C terminus 1D four epitope.
After incubating for 12 hours at four degrees Celsius, wash the resin three times as described in the text protocol. Next, label the receptor immobilized on the immuno affinity matrix by adding 0.1 millimolar fluorescein phosphine to a total volume of 0.3 to 0.5 milliliters incubate for 12 hours at room temperature before centrifuging following removal of the supernatant. Wash the resin to remove any unreactive label elute the labeled receptor with 100 microliters of Elian buffer by incubation on ice for one hour.
Resolve labeled samples by SDS page under reducing conditions. Wash gels briefly in PBS before labeling of a zeto fee. GPCR by Ingel fluorescence using a confocal fluorescence scanner with a 488 nanometer wavelength laser to detect receptor modified with fluorescein.
The unnatural amino acid mutagenesis methodology was used to C site specifically introduce molecular probes into A-G-P-C-R using the amber codon suppression technology. The photo cross-linking technique can then be used to investigate the structural aspects of GPCR ligand complexes in live cells, a radioactive labeled small molecule ligand tritiated. Ravi Rock was used to identify its binding site on the G-P-C-R-C-C-R five.
In this example, UV irradiation on cells expressing zeto fee. CCR five variance pre incubated with tritiated Ravi. Rock results in covalently cross-linked complexes that can be identified using the sensitive radioactive tag on the ligand.
Amongst the several positions on CCR five, tested lucine 28 zeto fee and tryptophan 86 zeto fee, were able to photo crosslink to tritiated Ravi Rock as depicted with significantly higher scintillation counts. The wild type receptor showed no cross linking to the radioactive ligand. Bio orthogonal labeling chemistries can be used to C site specifically modify a zeto feed.
GPCR variance one strategy involves the targeted epitope tagging of A-G-P-C-R in live cells using a peptide conjugated label flag peptide. Tri AAL PHOSPHINE labeling reagent is used to cite specifically and selectively modify a zeto fee. CCR five variance shown here is representative data from a sensitive and multi-step EISA detection strategy used to identify positions that are tagged with the flag peptide.
Amongst the positions on CCR five investigated all zeto fees. CCR five variants were expressed at the cell surface and approximately half of them successfully underwent targeted epitope tagging wild type CCR five served as a negative control for epitope tagging exhibited by the lack of Anti-Flag signal. In an alternative strategy detergent solubilized zeto feed G PCRs immobilized on an immuno affinity matrix such as anti 1D four monoclonal antibody conjugated to Spheros two B resin were labeled with a fluoro four conjugated label.
The Ingel fluorescence image demonstrates the successful fluorescent labeling of the GPCR opsin at three different positions as expected, the wild type opsin showed no background labeling as seen by the absence of a fluorescent band at the expected molecular weight of the receptor. While attempting this procedure, it's important to remember to confirm the full lens expression of the target GPCR with a set specific introduced acid AOL Alanine. After its development.
This technique will pave the way for researchers in the field of receptor biology to explore any receptor ligand complex or label a target receptor. Using the site specifically introduced unnatural amino acid, p zeto phenol alanine as a reactive probe. After watching this video, you should have a good understanding of how to use unnatural amino acid neogenesis to introduce a molecular probe into A-G-P-C-R to facilitate structural and dynamic studies of GPCR signaling complexes using targeted photo crosslinking and bio orthogonal labeling techniques.
