The overall goal of this procedure is to demonstrate a general technique to study ligand macromolecule binding using isothermal titration calorimetry, or ITC. This is accomplished by first preparing ligand and macromolecule solutions that are free of impurities and matched in both buffer composition and pH. The final concentrations of the ligand and macromolecule solutions are then determined.
Next ITC experimental parameters are set up followed by data collection. The final step of the procedure is to analyze and fit the data. Ultimately results showing ligand macromolecule interactions are obtained through graphical analysis of the titration data to determine the binding and thermodynamic parameters.
The main advantage of this binding technique over existing methods such as absorption and fluorescence titrations is that the enthalpy and thus the entropy of binding, can be determined from the same experiment. Sample preparation begins with the determination of proper concentrations for both the macromolecule and the ligand. A critical step to obtain accurate ITC fits information to consider while calculating these concentrations can be found in the written procedure.
In this demonstration, the binding of the co-factor N-A-D-P-H to the e coli chromosomal dihydro folate reductase enzyme is investigated. First reconstitute the lyophilized macromolecule in buffer to the desired concentration, then dissolve the ligand in the necessary volume of buffer. Next, check that the pH values of the macromolecule and the ligand samples are within plus or minus 0.05 pH units of the buffer and each other centrifuge, both the chromosomal dihydro folate reductase and N-A-D-P-H solutions in micro centrifuge tubes for five minutes.
At 8, 000 to 14, 000 RPM, this will pellet any particles in the solution, which can cause artifacts in the baseline of the ITC thermogram. Following centrifugation. Carefully check the concentrations of the macromolecule and ligand solutions by uves spectroscopy and record their exact concentrations.
Then make up the appropriate volume of each ITC solution by diluting the prepared stock macromolecule and ligand solutions to the necessary experimental concentrations. Finally, degas the samples to avoid signal artifacts due to air bubbles or the release of dissolved gases during the titration prior to loading the macromolecule and ligand into the ITC. Clean the sample cell and injection syringe according to the manufacturer's protocol.
Then rinse the sample cell two or three times with 1.8 milliliters of distilled water using a Hamilton syringe. Take care while handling the syringe as the long tip is easily bent. Next, rinse the sample cell several times with the same volume of buffer.
Once the sample cell is clean load 1.8 milliliters of the macromolecule solution into the cell. Being careful to avoid bubble formation at the point when the solution appears out of the cell stem stop sample addition. Remove any air bubbles using the Hamilton syringe by gently moving the needle up and down the sides of the cell to knock bubbles that are present.
Withdraw the syringe and remove any excess volume from the well at the top of the cell. Next, fill the reference cell with distilled water. Then attach a plastic syringe to the fill port of the injection syringe Using tubing.
Open the fill port on the VP viewer 2000 console. Rinse the injection syringe with distilled water, followed by a buffer rinse. Ensure the injection syringe is completely evacuated by drawing air through the system.
Place the needle of the injection syringe into the ligand solution and draw the solution into the syringe until entirely full. Continue drawing a little excess volume into the port and detach tubing following immediate closure of the fill port. Detach the tubing and plastic syringe purge and refill the injection syringe twice to eliminate any bubbles from the syringe.
Remove the injection syringe from the ligand solution and wipe off any drops on the side with a chem wipe. Be careful not to touch the syringe tip to the chem wipe or knock the syringe as this may cause a loss of volume from the syringe. Next, carefully place the injection syringe into the sample cell.
Set up the parameters for running the ITC using the V VP viewer 2000 program supplied by Micro Cal for binding systems. With strong heat signals such as the chromosomal DIHYDROFOLATE reductase N-A-D-P-H system, a large number of low volume injections will give more data points for fitting. Conversely, for systems that have weak heat signals, it is preferable to use a small number of large volume injections.
For the first one or two injections, set the injection volume to two microliters. These injections will eventually be discarded as they may give spurious results due to mixing between the ligand and macromolecule solutions. When inputting the time between injections, allow several minutes for the system to equilibrate and the heat signal to return to baseline.
Then choose the temperature of the experiment and input the stirring speed of the syringe necessary for adequate mixing of the ligand and macromolecule during the titration following experimental parameter setup, initiate the experiment. Once the experiment has finished, the final heat signal should be close to saturation. Clean the ITC according to the manufacturer's protocol, and repeat the titrations at least one or two more times to get reproducible data.
Finally, run a control where the ligand is titrated into buffer in the sample cell to determine the heat of dilution for the ligand fitting of the data can be easily performed using macros in any data fitting program. Origin seven supplied by micro Cal is used to demonstrate data fitting here. To begin load the first data file.
Check the raw thermogram for any sign of air bubbles or other artifacts in the signal if there are any artifacts such as spikes in the baseline or peaks in the absence of an injection point. Note these data points as they should be removed. Next, load the ligand dilution control data and subtract it from the binding isotherm at this time.
Remove all spurious data points, including the first few data points of the titration where dilution artifacts typically occur. Then select the data fitting model. Once the model is selected, the data can be fit by manually inputting initial guesses of the fitting parameters, including stoichiometry, enthalpy, and binding affinity.
However, if prior knowledge of any parameters is known from orthogonal experiments, these values should be entered as they will help avoid the data from becoming trapped in local mini during fitting. Once the data have been fit for the first titration, repeat the data analysis steps for the rest of the isotherms. Alternatively, the data can be fit globally using a program such as said fat, begin a global fit by saving the data in origin as a dot dat file.
Then import the data into said fat. Choose a model for fitting and globally fit the data switch back and forth between simplex and Mark HAR Leven fitting options until the global chi-squared value has reached a minimum. The representative ITC procedure demonstrated in this video monitors the binding of N-A-D-P-H to e coli chromosomal dehydro folate reductase.
The exothermic binding of N-A-D-P-H to de hydro folate reductase is evident in the raw thermogram as heat is released. Decreasing heat signals upon each successive injection indicates saturation of binding, which occurs within the first half of the titration. The purity of the components and good match of the buffers is noted from the saturated portion of the plot where there are only minimal heat signals arising from the heat of dilution of the ligand.
The data were fit to a single site model using the origin software to reveal the binding isotherm Two datasets were then fit to a single site model using said fat. The small values and random pattern of the residuals indicate that the thermodynamic parameters are reasonable. These parameters are also validated by comparison to literature values obtained by orthogonal methods.
After watching this video, you should have a good understanding of how to study ligand macro mo binding, using a VP ITC from micro by preparing solutions properly, setting up experimental parameters and analyzing the data.
