The overall goal of this procedure is to identify the correct physical model describing the binding of a macromolecule to its ligand and to determine the associated thermodynamic parameters. This is achieved by first preparing a stock solution of the macromolecule through extensive dialysis. The ligand of interest is then dissolved in the final dialysis buffer and the concentrations of all the components are determined in the second step.
The macromolecule and ligand samples are carefully diluted to a series of different concentrations. Next, a series of isothermal titration calorimetry, or ITC experiments are performed at several concentrations of both ligand and protein to produce a series of ITC Isotherms in the final step of the procedure. Global analysis of the series of experiments are performed using different physical models and the model giving the best overall agreement is identified as correct.
Today I'll be showing you a method that can help answer biophysical questions such as how protein molecules bind their ligands and can provide accurate measures of binding parameters such as entropy, entropy, and association constants Prior to the start of this protocol. Purify the protein of interest the macromolecule used in this demonstration is a immuno GLYCOSIDE N six prime, a subtle transferase one I or a C six prime one I.To begin, prepare four liters of dialysis buffer and cool to four degrees Celsius. Next dialyze a small volume of a macromolecule solution.
In this case, five milliliters of 400 micromolar AAC C six prime, one eye in 1.3 liters of dialysis buffer at four degrees Celsius. This can be done in a cold room or a refrigerator exchange with fresh buffer every eight hours and dialyze a total of three times. Save the final dialysis solution.
Filter 750 milliliters of the final dialysis solution through a 0.45 micron cellulose filter and store at four degrees Celsius. This buffer now referred to as the running buffer will be used for rinses and as a diluent. For the samples, thoroughly rinse a 0.2 micron syringe filter with running buffer.
Apply the protein sample and filter. Gently collect the sample in a conical tube prior to storing the protein solution, determine the protein concentration with a standard protein assay. In this case, the protein concentration is measured by ABSORBENCE at 280 nanometers and the enzyme concentration is calculated.
Using an extinction coefficient generated from the EXI proteomic server, store the protein in a manner to maximize long-term stability. AAC C six prime one eye must be stored at four degrees Celsius as it does not retain activity after freezing and thawing next dissolved the ligand and running buffer. For this demonstration, four milligrams of acetyl coenzyme A was dissolved in 200 microliters of running buffer to make a 25 millimolar stock solution.
This ligand can be stored at minus 78 degrees Celsius until ready to use to prepare the ITC samples, first, calculate the concentration of enzyme to obtain a C value of 64 as described in the written protocol for AAC six prime one I.This corresponds to a concentration of 192 micromolar. Prepare a two milliliter solution of the macromolecule at this concentration, diluting the stock solution with the filtered running buffer FIA settle co-enzyme a thaw, the acetal coenzyme a solution in an ice water bath. Once the acetal co-enzyme a solution is thawed mix, gently calculate the concentration of ligand solution by multiplying the macromolecule concentration by the number of binding sites multiplied by 10.
Prepare the acetyl co-enzyme a solution by adding 80 microliters of the 25 millimolar ACE co-enzyme, a stock solution to 420 microliters of running buffer and mixing. Transfer the diluted acetyl co-enzyme ACE solution into a pipette filling tube and return the remaining 25 Millimolar stock solution to minus 78 degrees Celsius for storage. Degas the a C six prime one eye and a subtle co-enzyme, A solutions under vacuum for five minutes at 19 degrees Celsius, which is one degree Celsius below the desired experimental running temperature.
To set up the injection syringe, insert the syringe through a syringe holder until the prem mounted syringe clamp is at the same height as the holder. Feed the second syringe clamp over the injection syringe until it's firmly pressed against the bottom of the syringe holder. Gently tighten the clamp with the provided 0.05 inch ballpoint hex drive.
Next, place the syringe holder into the pipette holder. Gently slide the pipette injector into the injection syringe and make sure the plunger tip is fed directly into the hole of the syringe. Once fully inserted, screw the locking collar of the syringe holder into the pipette injector.
Before loading the A a C six prime one eye protein sample solution, use the cell washing system provided with the instrument to wash the sample cell with at least 50 milliliters of running buffer. Remove the residual buffer from the sample cell by aspiration with a long needle 2.5 milliliter glass syringe. Remove the solutions from the degassing vacuum using a second clean dry long needle.
2.5 milliliter syringe. Carefully draw a minimum of 1.8 milliliters of the A a C six prime one eye protein sample into the syringe. Use care to avoid air bubbles.
Once the syringe is loaded with the protein sample, insert the needle in the sample cell and gently touch the bottom of the cell. Raise the needle approximately one millimeter and gently inject the sample into the cell until excess liquid is visible above the top of the sample cell. To flush the cell of any trapped air bubbles, raise the needle by about one centimeter while ensuring solution remains in the overflow and quickly withdraw and inject approximately 0.25 milliliters of the solution.
Now gently slide the needle upwards along the side of the overflow. In the sample well the needle will hit a ledge. Withdraw all of the liquid above this ledge as the ledge indicates the desired height for the running solution.
To load the injection syringe, attach the plastic tube bloating syringe onto the fill port and lower the plunger tip to the top of the fill port. Now place the pipette filling tube of DGAs ligand solution at the bottom of the pipette holder. Check that the syringe tip does not touch the bottom of the filling tube and slowly draw the solution into the syringe until a small amount enters the tube of the loading syringe.
Next, close the fill port by lowering the plunger tip. This is done by clicking on the close fill port button. Then to remove any air bubbles that may have become trapped during loading.
Purge and refill by clicking the purge refill button. Purge and refill a total of three times. Once the purge refill process is complete, remove the filling tube from the pipette holder and gently wipe the syringe tip with lab tissue paper.
Now lower the syringe of the pipette assembly into the sample. Well use caution and proceed slowly as the needle can easily bend. Ensure the syringe is completely inserted by pressing down on the base of the locking collar.
Once the syringe is secured, set the desired running temperature and select a reference power that is slightly greater than the maximum injection heat flow expected. In this example, 20 degrees Celsius and 20 micro calories per second are used as the running temperature and reference power respectively. Then program the desired injection volumes and delays.
Here, 28 injections are used with the first having a volume of two microliters and a 62nd delay and subsequent injections having a volume of 10 microliters with 332nd delays. The experiment demonstrated here provides the foundation to generate a dataset at a single concentration to generate the additional curves necessary. Repeat this process with decreasing macromolecule and ligand concentrations.
Be sure that all macromolecule and ligand samples originate from the same stock solution to minimize error in concentrations. Shown here is the raw isothermal titration calorimetry trace produced from the titration of 3.86 millimolar ligand acetyl coenzyme A into 192 micromolar of the micro molecule. A A C six prime one.
Eye integrated values were used to determine the binding parameters with a two site sequential fit. This graph depicts the isotherms generated by ITC of varied concentrations of ACE Coenzyme A and AAC C six Prime One Eye. The experimental data represented by open circles were fit to both a two sets of sites independent model, and a two site sequential model.
Better agreement is seen with the two site sequential model. The protein concentrations used are defined in the accompanying text. When performing this procedure, it is important to be very accurate with your dilution as the change in concentrations can have a drastic impact on your results.
