The overall goal of this methodology is to extract the thermodynamic parameters governing biomolecular folding and binding interactions by differential scanning calorimetry, or DSC, using a thermolabile ligand in as little as two experiments. This method can help answer key questions in the biocalorimetry and drug design fields such as what are the relative magnitudes of the thermodynamic parameters governing interactions between biomolecules and tight binding inhibitors? The main advantage of this technique is that a full ligand titration is performed in a single experiment permitting rapid extraction of thermodynamic parameters from the global fitting analysis.
To begin, prepare buffers for dialysis of the purified biomolecule into solution of ligands. Dialyze the biomolecule against at least one liter of buffer using dialysis tubing with 0.5 to 1.7 kilodalton cutoff. The most important step in obtaining high-quality DSC data is the dialysis of the biomolecule ligand against the desired buffer.
This ensures that there are no buffer mismatched artifacts present in the DSC data. Filter the final buffer which is referred to as the working buffer through a 0.2-micron filter that has been thoroughly equilibrated with buffer. Weigh out the desired masses of the ligands and dissolve them in filtered working buffer.
If the desired ligand concentrations require masses that are too small to accurately weigh, make a concentrated ligand stock solution. Filter the biomolecule stock solution through a 0.2-micron filter that has been thoroughly equilibrated with working buffer. Then, determine the biomolecule concentration by absorbance measurements.
Store the prepared biomolecule and ligand in a four degrees Celsius refrigerator or at minus 20 or minus 80 degrees Celsius if the biomolecule and ligands tolerate freezing and long-term storage is required. Degas the buffer by a molecule and ligand solutions in a tabletop degasser prior to loading into the differential scanning calorimeter, or DSC. Unscrew the pressure handle from the DSC.
Then, run silicon tubing from the working buffer and attach it to the front flange of the reference capillary. Create a bridge between the reference and sample capillaries by connecting the rear reference flange to the front simple flange. Next, attach a piece of silicon tubing to the rear sample flange that runs to a waste flask with a vacuum line attached.
Turn on the vacuum line to flush the DSC with 200 milliliters of working buffer. Begin by attaching roughly three to five-centimeter sections of silicon tubing to the reference capillary flanges. Next, insert a one-milliliter pipette tip into the silicon tubing of the rear flange.
Draw 0.8 milliliters of working buffer with a pipette and insert the pipette tip with buffer into the silicon tubing of the front reference flange. Gently press the pipette plunger down to pass the working buffer through the front silicon tubing into the reference capillary and up into the attached pipette tip of the rear flange. Press the pipette plunger down until the working buffer level reaches just above the front silicon tubing, then release the pipette plunger until the working buffer level reaches just above the rear silicon tubing.
Continue passing the working buffer back and forth in the reference capillary to purge the volume of bubbles. Next, cap the rear pipette tip with a forefinger and gently pull up on the rear pipette tip and front pipette to remove them from the reference flanges with the silicon tubing attached. Load the sample capillary with working buffer as before.
Place a black plastic cap on the rear reference sample flanges leaving the front flanges uncovered. Attach the pressure handle to the DSC and then open the DSC software and pressurize the instrument by clicking the red up arrow at the top of the interface once the power reading has stabilized. The DSC power is indicated in a box at the top right of the interface along with the instrument temperature and pressure reading.
Equilibrate the DSC with working buffer by performing a forward and reverse scan in the experimental method tab on the left side of the screen. Ensure that the scanning option is selected to run the DSC and temperature scanning mode. In the temperature parameters inset under the experimental method tab, click the button for heating.
Enter one and 100 degrees Celsius for the lower and upper experimental temperatures, one degree Celsius per minute for the scan rate and 60 seconds for the equilibration period. Click the AddSeries button under the input field for the equilibration period. Enter two into the steps to add field in the popup window and check the Alternate Heating/Cooling box.
Click OK.The added scans appear in the lower portion of the interface. Check that the parameters for each scan are as desired. Start the experiment by clicking the green play button at the top of the interface.
Navigate to the desired window and input a file name for saving the experiment in the popup window. View the experiment progress by clicking the data tab to the right of the experiment method tab. Run reference experiments for a baseline subtraction of the sample data by reloading the DSC with working buffer in both capillaries.
Collect multiple forward and reverse scans over a suitable temperature range at one degree Celsius per minute with an up equilibration time of 120 seconds. Delete the previous buffer equilibration scans from the lower portion of the interface by highlighting each individually and clicking the red X to the middle right of the interface. Add the new scans by clicking the AddSeries button entering 20 in the field for steps to add and checking the Alternate Heating/Cooling box.
Then, click OK and run the experiment by clicking the green play button as before. Repeat these steps with working buffer containing the desired concentration of ligand in both capillaries to obtain the reference experiments for the ligand. Collect two separate experiments to be used in acquiring the rate constant for thermolabile conversion.
For the free biomolecule data set, ensure that the reference capillary contains the working buffer while the sample capillary contains the free biomolecule at the desired concentration in working buffer. Next, run the sample experiments using the same DSC loading procedure and experimental parameters used in the reference scans. For the ligand-bound experiments, ensure that the ligand is in the working buffer and the reference capillary and the biomolecule plus ligand are in the working buffer in the sample capillary.
Flush the system between additions of different ligands as before. Perform one additional experiment with the biomolecule bound to the thermolabile ligand where the high-temperature equilibration period is increased to 600 seconds and all other experimental parameters are the same as before. Proceed to the data processing and analysis as described in the text protocol.
The position and height of the thermolabile ligand bound peak successively shifts down towards that of the unbound molecule as the thermolabile ligand is depleted with each scan. The 3D maturation profile is used as a reference for the endpoint of thermolabile ligand conversion. Increasing the high-temperature equilibration period yields more pronounced reductions of the thermolabile ligand concentration with each scan relative to the short equilibration period dataset.
Using the optimized global fit concentration parameters from the two datasets, the rate constant for ligand conversion at the high equilibration temperature can be calculated. Once mastered, this technique can be done in 72 hours if performed properly. When attempting this procedure, it is important to remember to degas the biomolecule ligand as well to purge the DSC capillaries of bubbles using the loading pipette to prevent data artifacts.
After watching this video and reading the article, you should have a good understanding of how to perform DSC experiments on biomolecules as well as the implementation of global fitting analyses in order to rapidly extract thermodynamic parameters governing folding and binding interactions. Though this method can provide insight into equilibrium folding binding interactions, it can also be applied to other systems such as biomolecules that exhibit slow-folding and/or ligand-binding kinetics.
