Our method helps study the kinetics of a protein complex formation. It describes how tight a protein complex is, and if the protein complex formation is interfered by other proteins. This technique enables studying protein-protein interaction in quantitative way using fluorescence as a reporter, our assay can monitor the association and disassociation of a protein complex in real-time.
To begin, design the FRET assay starting with the data for the COL1 CAND1 complex from the protein database. Load the structure in PyMOL, and then go to the wizard menu and use the measurement function to estimate the distance between the first amino acid of CAND1 and the last amino acid of COL1. Then, load the online spectra viewer and view the excitation and emission spectra of 7-amino-4-methylcoumarin and FlASH simultaneously.
AMC is the FRET donor and FlASH is the FRET acceptor. Prepare cells with the FRET donor protein by following along in the accompanying text protocol. Then, purify the COL1 sortase RBX1 complex by first adding 50 milliliters of lysis buffer to a pellet of E.Coli cells expressing the COL1 sortase RBX1 complex.
Ice the cells on ice with sonication at 50%amplitude. Alternate the power for three minutes so that it is one second on, and then one second off to prevent overheating the sample. Transfer the resulting cell lysate into a 50 milliliter centrifugation tube and remove the cell debris by centrifugation at 25, 000 times g for 45 minutes.
Then, add the supernatant to five milliliters of glutathione beads and incubate them at four degrees Celsius to clear the cell lysate. Following incubation, centrifuge the bead lysate mixture at 1, 500 times g for two minutes at four degrees Celsius. Then, remove the supernatant.
Next, wash the beads with 5 milliliters of lysis buffer having no protease inhibitor. After centrifuging the solution, remove the supernatant. Now, add three milliliters of lysis buffer to the washed beads and transfer the bead slurry into an empty column.
To the new column, also add five milliliters of elution buffer and then incubate the column for 10 minutes and collect the eluate. Next, add 200 microliters of thrombin at five milligrams per milliliter to the eluate from the glutathione beads. Following an overnight incubation at four degrees Celsius dilute the protein sample three fold with buffer A.Load the protein sample to a buffer A equilibrated cationic exchange chromatography column at zero point five milliliters per minute.
Then, add a gradient of zero to 50%of buffer B in buffer A to elute the protein with a flow rate of 1 milliliter per minute. Next, pool the eluate fractions containing the FRET donor protein and concentrate it down to two point five milliliters using an ultrafiltration membrane with a 30 kilodalton cutoff. Now, add 7-amino-4-methylcoumarin to the c-terminus of COL1 through sortase mediated transpeptidation by first changing the buffer in the FRET donor protein sample into the sortase buffer using a desalting column.
To accomplish this, first equilibrate a desalting column with 25 milliliters of sortase buffer. Then, load two point five milliliters of the FRET donor protein solution into the column and discard the flow through. Next, elute the sample with three point five milliliters of sortase buffer and collect the flow through.
In 900 microliters of the eluate, add 100 microliters of 600 micromolar purified sortase A solution and 10 microliters of a 25 millimolar GGGG-AMC peptide. Incubate the reaction mixture at 30 degrees Celsius in the dark overnight to generate COL1-AMC-RBX1. Now, load all of the sample onto the size exclusion chromatography column and elute it with one point five times the column volume of buffer.
In the end, pool the eluate fractions containing COL1-AMC-RBX1. Follow along in the text protocol to prepare the FRET acceptor protein. Many of the steps are similar to the FRET donor protein preparation.
Once finished, prepare the FlASH CAND1 solution. First, add one microliter of the FlASH solution to 50 microliters of the freshly prepared tetra-cys-CAND1 solution. Mix the combined solutions well and incubate the mixture at room temperature in the dark for one to two hours to form the FlASH CAND1 solution.
In 300 microliters of FRET buffer, add COL1-AMC-RBX1 to a final concentration of 70 nanomolar and transfer the solution into a cuvette. Place the cuvette in the sample holder of a fluoriometer. Excite the sample with 350 nanometer excitation light and scan the emission signals from 400 to 600 nanometers at one nanometer increments.
Then, in 300 microliters of FRET buffer, add both COL1-AMC-RBX1 and FlASH-CAND1 to a final concentration of 70 nanomolar. Excite the sample with 350 nanometer excitation light and scan the emission signals from 400-600 nanometers at one nanometer increments. Set the excitation light at 350 nanometers and use a band pass filter that allows 450 nanometers emission light to pass and blocks 500 to 650 nanometer emission light.
With the sample valves at the fill position connect a three milliliter syringe filled with water. Then, wash the two sample syringes with water by moving the sample syringe drive up and down several times, discarding the water when finished. Next, connect a three milliliter syringe filled with FRET buffer.
Wash the two sample syringes with the FRET buffer by moving the sample syringe drive up and down several times, discarding the water when finished. Now, connect a three milliliter syringe and load the first sample syringe, syringe A, with 100 nanomolar COL1-AMC-RBX1 in the FRET buffer. Then turn the sample valve to the drive position Also, connect a three milliliter syringe to syringe B and load syringe B with 100 nanomolar of FlASH CAND1 in the FRET buffer and turn its valve to the drive position.
Open the control panel under acquire in the software and record the emission of COL1-AMC over the course of 60 seconds. Then, take a single shot. Fit the decrease and the fluorescent signals measured over time from each shot to a single exponential curve.
Wash the system as previously shown and then add a solution of 100 nanomolar COL1-AMC-RBX1 and 100 nanomolar FlASH CAND1 in the FRET buffer into syringe A.Connect it to the system while it is in the fill position Load syringe A and then turn the sample valve to the drive position. Next, load a solution of Skp1-Skp2 into syringe B.Connect it to the system while it is in the fill position. Load syringe B and then turn the sample to the drive position.
In the software, go to acquire and open the control panel. Set up the program to record the emission of COL1-AMC over 30 seconds. Then take a single shot.
The fluorescence signals increase over time after mixing solutions from syringe A and syringe B.When 70 nanomolar each of COL1-AMC and FlASH CAND1 were mixed to generate FRET, two emission peaks were present in the emission spectra. And the peak of COL1-AMC became lower, and the peak of FlASH CAND1 became higher. When the full length CAND1 was used for FRET the donor peak showed a 10%reduction in intensity.
However, when CAND1 with its first helix truncated was used the reduction of donor peak intensity was increased to 30%suggesting higher FRET efficiency To confirm that the signal changes were resulted from FRET between COL1-AMC and FlASH CAND1, the donor FRET was mixed with excess unlabelled CAND1 known as the Chase in the acceptor. As a result the donor peak was fully restored and the acceptor peak was decreased. Shown here is a plot of the average observed K value with the CAND1 concentration following linear regression analysis.
To measure the on constant of the complex, 50 nanomolar COL1-AMC was used in a series of observed dissociation rate constants were measured by mixing 50 nanomolar COL1-AMC with increasing concentrations of FlASH CAND1. Similar to the measurement of the on constant, the observed dissociation constant of COL1 and CAND1 was found by monitoring the restoration of the donor signal over time on the stopped flow fluorometer. Here the donor signal increased quickly and it revealed an observed K value of zero point four per second.
In contrast, when buffer was added to the pre assembled complex, no signal increase was observed suggesting the fast dissociation of COL1 CAND1 was triggered by Skp1 Skp2. Make sure to minimize the amount of light the donor and acceptor proteins are exposed to. Try to keep fluorophores in the dark as much as possible.
