I was wondering someone might have any idea what could cause the result we observe below. We are studying binding between a protein and small molecule ligand. In order to determine the enthalpy of solvent reorganization at the binding site, we follow the technique described in this paper: Article A Direct Measure of the Contribution of Solvent Reorganizati...
, where ITC is performed in one experiment with protein and ligand in aqueous solution, and in a second experiment identically but using D2O. Sample prep: Paired H2O and D2O experiments used the same protein stock solution. Half the protein stock was dialyzed into the D2O buffer. The titrant was the ligand diluted >1000X into the dialysis buffer. This buffer was prepared by lyophilizing the H2O buffer and adding the same volume of D2O. This was repeated four times with two different protein preps, and always resulted in curves like the ones shown here. The instrument is a MicroCal iTC200.The ITC results look as expected for the aqueous solution (exothermic, first figure below), but in the deuterated solution we unexpectedly observe what seems to be endothermic binding (second figure below). One would expect both results to look similar.
Any ideas what could cause this?
First of all, I would repeat the experiments, just in case.
And I would perform control experiments by injecting ligand into buffer.
If everything goes well... then:
Protein-ligand interaction results in release of water molecules from the binding interfaces to the bulk solution. Water molecules located onto hydrophobic binding sites do not interact well with the reactant molecule, establishing stronger water-water hydrogen bonds than those in the bulk solution. Therefore, the dehydration accompanying the protein-ligand binding, besides the large gain in entropy, contributes with an unfavorable enthalpic contribution to the overall interaction enthalpy.
D2O establish stronger hydrogen bonds than H2O, and D2O is more polar than H2O. However, as it occurs with H2O, the key point is the difference in hydrogen bond strength between D2O molecules hydrating the binding interfaces and that of D2O in the bulk solution; this difference will determine the magnitude of the dehydration enthalpy contribution to the overall interaction enthalpy.
Anyway, I am not an expert in isotope effects in water.
Dear Amanda,
Upon reading multiple times the description of your experiments, I can only surmise that something went wrong during the lyophilization process, whereby the ligand solution is not fully balanced with the protein solution in the D2O run. Since you have dialyzed the protein in D2O buffer, couldn’t you simply dissolve the ligand in the final dialysate? Unless I am missing something, I think this might help in eliminating the high background heats observed in the D2O run. As recommended by Adrian, the ligand into buffer run will be crucial in this case.
Good luck with you ITC measurements.
Yes, check the recommendation by Conceicao Minetti.
On the other hand, because buffer ionization can contribute to the observed enthalpy through its ionization enthalpy if protons are released or uptaken upon complex formation, if the ionization properties of the buffer molecule (in particular, the ionization enthalpy) are different in D2O compared to those in H2O, the contribution of the buffer to the observed enthalpy might be different in D2O compared to that in H2O.
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