Open-ITC: an alternate computational approach to analyze the isothermal titration calorimetry data of complex binding mechanisms.
Isothermal titration calorimetry (ITC) is an important technique used in quantitatively analyzing the global mechanism of protein-protein or protein-ligand interactions through thermodynamic measurements. Among different binding mechanisms, the parallel and ligand induced protein oligomerization mechanisms are technically difficult to analyze compared with a sequential binding mechanism. Here, we present a methodology implemented as a program "Open-ITC" that eliminates the need for exact analytical expressions for free ligand concentrations [L] and mole fractions of bound ligand θ that are required for the thermogram analysis. Adopting a genetic algorithm-based optimization, the thermodynamic parameters are determined, and its standard error is evaluated at the global minimum by calculating the Jacobian matrix. This approach yielded a statistically consistent result for a single-site and a two-site binding protein-ligand system. Further, a comparative simulation of a two-step sequential, a parallel, and a ligand induced oligomerization model revealed that their mechanistic differences are discernable in ITC thermograms, only if the first binding step is weaker compared with the second binding step (K(1) <K(2)). We find this to be valid even for the cases where the enthalpies of each of the binding process did not vary significantly.
Department of Chemistry and Biochemistry, Auburn University, Auburn, AL 36849, USA.
Janarthanan
J
Krishnamoorthy
Smita
S
Mohanty
D000465:Algorithms; D002151:Calorimetry; D003198:Computer Simulation; D003568:Cytidine Monophosphate / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D008956:Models, Chemical; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*; D012260:Ribonucleases / Q000737:chemistry; D013816:Thermodynamics
D008024:Ligands; D011506:Proteins; D012260:Ribonucleases; D003568:Cytidine Monophosphate