Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

The overall goal of this procedure is to estimate the dissociation constant for a protein ligand interaction. This is accomplished by first preparing mixtures of the protein with different concentrations of the ligand together with an indicator dye. The second step is to heat these samples whilst recording the fluorescence of the indicator to monitor the unfolding of the protein.

Next, the protein unfolding curves are converted to a series of melting temperatures. The final step is to fit these data to an appropriate model to estimate the dissociation constant. Ultimately, differential scanning fluorometry is used to better understand the interaction of a protein with its ligands.

The main advantage of this technique over existing methods like isothermal, titration, calorimetry, and surface plasma resonance is that this method can be completed within a few hours using only moderate quantities of sample in an instrument that is already available in most institutes. This method can help answer key questions in the protein chemistry field, such as the affinity of ligands for proteins and the relative strength of interactions. Generally, individuals new to this method would struggle because the choice of samples to use and analysis of the data can both be difficult.

Prior to starting this procedure, prepare the following solutions. A mixture containing the reagents detailed in this table and stocks of the ligand of interest at the highest available concentration, as well as six tenfold dilutions of this concentration. If an approximate dissociation constant is known from prior data, prepare at least two concentrations above and below the kd.

An example table is shown here. Aliquot 18 microliters of the mixture into each of eight wells. In A-Q-P-C-R plate, add two microliters of solvent to the first well to each of the remaining seven wells, add two microliters of each member of the ligand dilution series.

Place A-Q-P-C-R seal over the plate to achieve a good seal of the plate. Place a hand applicator in the middle of the plate, smooth down the seal to one side, and then repeat on the other half of the plate. Centrifuge the plate at 500 times G for two minutes to remove air bubbles.

Next, place the plate in a step one QPCR instrument. Select the melt curve option the rocks filters, and choose the fast ramp speed. Run a thermal denaturation at the conclusion of the instrument run.

Click on the analyze button on the screen. Save the result file. Open the protein thermal shift software.

Create a new study in the properties tab. Give it a name, and in the conditions tab, detail the ligands. Move to the experiment files tab and import the saved results file.

Set the contents of each. Well move to the analysis tab and press the analyze button to analyze the results. Check that the protein in the presence of solvent alone gives a result similar to that shown in this figure.

Next, examine the melting temperatures observed in the results in the replicate pain. Ensure that this shows a clear increase in melting temperature with increasing ligand concentration. These data will be used to provide an approximate value for the dissociation constant as described in the accompanying paper.

These solutions are required for the procedure to determine the dissociation constant, A master mix as detailed in this table and stocks of the ligand at 15 different concentrations, which will be diluted tenfold. In the final experiment. Ideally include ligand concentrations, at least two orders of magnitude above and below the estimated kd, and center the concentrations on the estimated kd.

An example dilution series is shown here. Focus on seven of the points within an order of magnitude of the estimated KD with another four points on either side of this. If there is a choice, include more points at values that are saturating.

Add 120 microliters of the master mix to each of eight wells in a U bottomed 96 well plate to act as a reservoir for convenient dispensing of the master mix. Then use an eight channel pipette to dispense 18 microliters of the master mix into one column of a PCR plate. Repeat for a further five columns to give a total of 48 filled wells in a six by eight pattern on the plate.

Next, add 20 microliters of the ligand stalks or the solvent to each of eight wells. In another U bottomed 96 well plate Using an eight channel pipette aspirate two microliters of eight different ligand stocks or solvents, and add these to one column of the PCR plate that was filled with the master mix. Repeat with the same eight ligand or solvent stocks.

For two further columns, aspirate two microliters of the remaining eight ligand or solvent stocks, and add these to a fourth column in the plate. Repeat this for two further columns. This will give triplicate samples for all 16 ligand and solvent samples.

Place A-Q-P-C-R seal over the plate. Centrifuge the plate at 500 times G for two minutes. Place the plate in the QPCR instrument and run a thermal denaturation using the parameters specified earlier at the conclusion of the instrument run.

Click on the analyze button on the screen. Save the result file. Open the protein thermal shift software.

Create a new study in the properties tab. Give this a name and in the conditions tab, detail the ligands. Move to the experiment files tab, import the saved results file and set the contents of each.

Well move to the analysis tab and press the analyze button. Choose the replicates tab from the menu on the left hand side of the screen to show the results. As triplicates.

Assess the reliability of the data based on how tight the triplicates are. Should the triplicates show poor reproducibility. Examine the raw data closely.

Analyze the data using both the boltman or derivative methods. To assess the melting temperature, select the replicate results tab and in the replicate results plot, toggle the plot by button between tm, boltzmann and TM derivative. Select the method that gives the greater reproducibility for the sample.

For samples that show multiple transitions, it is almost always best to use the derivative method in multiple melt mode. Export the results for further investigation with Excel using the export tab. Begin this analysis by creating a table in Excel of the ligand concentrations and the melting temperature.

Open the GraphPad prism software and create an XY table. Enter the data using the X column for the ligand concentrations and the Y column for melting temperature results. In the analysis tab, select the nonlinear regression curve fit option.

To enter the correct model, select new and create new equation. Insert the appropriate equation as single site ligand binding. Select the rules for initial values box and enter rules for initial values.

Constrain the parameter P as constant equal to enter the final concentration of protein. Select analyze to perform the analysis, additional analysis to fit data to a cooperative model or to fit data to curves. Showing binary shifts in melting temperature are described in the accompanying protocol text.

This method was used to measure the interaction of hexokinase with glucose. An initial screen suggests a likely KD from 0.2 to 1.7 millimolar. The results of a larger screen show a good fit to the model for a single binding event with a KD of 1.2 plus or minus 0.1 millimolar, the putative HETO SQUA L transferase WCBM shows a strong thermal shift on binding to GTP.

An initial screen suggested a KD of 200 to 500 micromolar. A detailed experiment suggests an apparent KD of 120 plus or minus 20 micromolar. However, when a logarithmic scale is used for the X axis, there is a significant discrepancy between the model and data analysis of the same data with a cooperative model shows an excellent fit to the data, implying that WCBM is anti cooperative in its binding to GTPA rather unusual result is observed with the putative GDP 60, oxy Beta D mano, Heur two oh, ACE WCBI.

Without ligand, and at high ligand concentrations, a simple monophasic melting pattern is observed. However, at intermediate ligand concentrations, two distinct melting peaks are observed. The transition between the two sets of peaks is dose dependent across the full range of concentrations modeling of the biphasic melting.

As a sum of a proportion of the ligand, free and high ligand results gives a good fit to the data. This fit is improved by extrapolating the result observed for high ligand concentration to full occupancy. The data obtained for the proportion of WCBI bound to ligand shows an excellent fit to a simple binding model.

With a KD of 58 plus or minus two micromolar Once mastered, this technique can be done in four or five hours, including repeats if it is performed properly Following this procedure. Other methods like tiptop vein fluorescence and isothermal titration calorimetry can be performed in order to answer additional questions like temperature dependence, and STA geometry. After watching this video, you should have a good understanding of how to determine an estimate for the dissociation constant of an interaction using differential scanning flu.