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

Abstract

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.

ITC and SPR Analysis Using Dynamic Approach

“…The same parameters were used to simulate the thermograms of RNAHH (Fig (2 A,B)), FEOTF54 (Fig (2 C,D)), PROTDB (Fig (2 E,F)) or PERSSON (Fig (2 G,H)) using dynamic models such as (1) & Fig (3A,B). The model fitted well to the experimental data and the results were comparable to that of the previously reported values [28,29]. In our current analysis, because of large residual values in initial fits for BH3I-1 + hBCL XL , we let the injection volumes to be varied during the optimization.…”

“…Further, we have also analysed the experimental ITC data of 2'-CMP + RNASE and BH3I-1 + hBCL XL , and determined the kinetic and thermodynamic parameters of the binding process. The results were consistent with the earlier reports [27,28]. Dynamic approach based simulation of the SPR profiles for a single binding site also yielded accurate profiles similar to reported experimental profiles.…”

“…Based on this approach, all the optimized parameters were found to be invariably sensitive and significant to the models used in the analysis of RNASE and hBCLxl. Cals/mol) [28,29].…”

High-Precision Isothermal Titration Calorimetry with Automated Peak-Shape Analysis

“…Experimentally contributing factors often remain unclear, although bubbles, bent needles, and external mechanical or electronic events have been identified as sources of noise and poorly reproducible signals 9 .Unfortunately, techniques for the analysis of thermograms with imperfectly constant baselines have rarely been addressed in the scientific literature. Strategies such as a spline model or other smooth interpolation functions for long-term drifts 24,26,44 , as well as linear interpolation between average pre- and post-injection baselines 27 have been reported. The examples of Figure 1 and Figure S1 illustrate where these approaches would clearly not be detailed enough and introduce errors.…”

“…Although the importance of baselines has been recognized previously 25 , the problem of baseline assignment is only very sparsely addressed in the calorimetry literature 24,26,27 . To the best of our knowledge, the algorithms implemented by instrument manufacturers for default baseline assignment have not been entirely published and thus remain unclear.…”