ITC is a powerful tool for studying the binding of a ligand to its host. In complex systems however, several models may fit the data equally well. The method described here provides a means to elucidate the appropriate binding model for complex systems and extract the corresponding thermodynamic parameters.
1. Preparing stock solutions
2. Preparing ITC samples
3. Setting up the syringe14
4. Loading the sample cell14
5. Loading injection syringe and initiating run14
6. Subsequent Runs
7. Data Analysis
8. Representative Results
Representative data are shown in Figure 1. The shapes of the isotherms should vary with concentration. Sharper transitions are expected for higher c-values (i.e. higher protein and ligand concentrations) (Figure 2).
In the case of AAC(6′)-Ii, the two-site sequential model gives a better fit than one describing two sets of identical, independent sites with adjustable stoichiometries.
Figure 1. Isotherms produced by titration of AcCoA (3.86 mM) into AAC(6′)-Ii (192 μM). A) Raw ITC trace. B) Integrated values used for determining binding parameters (squares) with a 2-site sequential fit (-).
Figure 2. ITC isotherms for AcCoA titrated into AAC(6′)-Ii at varying concentrations. The experimental data (open circles) were fit to a 2-sets-of-sites independent model (dashed magenta) and a 2-site sequential model (solid blue). The 2-site sequentional model clearly gives better overall agreement. The concentrations employed were A) 6 μM, 0.25 mM, B) 12 μM, 0.25 mM, C) 24 μM, 0.5 mM, D) 48 μM, 1.0 mM, E) 96 μM, 1.9 mM, and F) 196 μM, 3.86 mM, for AAC(6′)-Ii and AcCoA respectively.
This analytical portion of variable-c fitting has been previously described in detail10. Here we report practical aspects of collecting variable-c datasets suitable for this approach. It is essential that all protein and ligand samples are drawn from the same stock solutions. Therefore it is important that sufficient stock solution is prepared initially to complete the entire series of experiments. This ensures the ratio of AAC(6′)-Ii and AcCoA is constant among all experiments, and reduces random fluctuations in concentration on a sample-to-sample basis.
In this case, much care must be taken in the handling of the ligand. AcCoA is chemically unstable in solution so it is essential that the stock solution remains liquid for very short periods of time. Once the appropriate amount is aliquoted from the stock solution, the stock must immediately be refrozen. If this is not done, then the AcCoA may degrade over time and the concentrations will not be correct in later runs. Once aliquoted, AcCoA samples must be used immediately. In the case of proteins that are unstable, similar precautions will be necessary.
This procedure can be easily modified to study other complex systems.10 The concentrations used must be adapted to the binding constants of the specific system. A wide range of c values between about 1 and 10008,10 is required across the dataset.
The authors have nothing to disclose.
This work was supported by the Canadian Institutes of Health Research (CIHR), National Science and Engineering Research Council (NSERC), and a CIHR training grant scholarship (to L.F.). We thank Prof. Gerard D. Wright (McMaster University, Canada) for the AAC(6)-Ii expression plasmid.
Material Name | Typ | Company | Catalogue Number | Comment |
---|---|---|---|---|
Acetyl coenzyme A (AcCoA) | Sigma-Aldrich | A2056 | ||
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) | Fisher | 7365-45-9 | ||
ethylenediaminetetraacetic acid (EDTA) | Sigma-Aldrich | 431788 | ||
Spectra/Por 2 Dialysis Tubing | Spectrum Labs | 132678 | ||
Sterile Syringe Filter (0.2 μm) | VWR | 281445-477 | ||
Cellulos Nitrate Membrane Filters (0.45 μm) | Whatman | 7184-004 | ||
VP-ITC | MicroCal | VP-ITC | Microcalorimeter used for measurements | |
ThermoVac | MicroCal | USB Thermo Vac | Temperature Controlled Degassing Station |