Open-ITC: an alternate computational approach to analyze the isothermal titration calorimetry data of complex binding...

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)