Strategies for assessing proton linkage to bimolecular interactions by global analysis of isothermal titration calorimetry data

Abstract

Isothermal titration calorimetry (ITC) is a traditional and powerful method for studying the linkage of ligand binding to proton uptake or release. The theoretical framework has been developed for more than two decades and numerous applications have appeared. In the current work, we explored strategic aspects of experimental design. To this end, we simulated families of ITC data sets that embed different strategies with regard to the number of experiments, range of experimental pH, buffer ionization enthalpy, and temperature. We then re-analyzed the families of data sets in the context of global analysis, employing a proton linkage binding model implemented in the global data analysis platform SEDPHAT, and examined the information content of all data sets by a detailed statistical error analysis of the parameter estimates. In particular, we studied the impact of different assumptions about the knowledge of the exact concentrations of the components, which in practice presents an experimental limitation for many systems. For example, the uncertainty in concentration may reflect imperfectly known extinction coefficients and stock concentrations or may account for different extents of partial inactivation when working with proteins at different pH values. Our results show that the global analysis can yield reliable estimates of the thermodynamic parameters for intrinsic binding and protonation, and that in the context of the global analysis the exact molecular component concentrations may not be required. Additionally, a comparison of data from different experimental strategies illustrates the benefit of conducting experiments at a range of temperatures.

SEDPHAT – A platform for global ITC analysis and global multi-method analysis of molecular interactions

“…However, the global analysis of multi-site interactions contains features that are independent of the concentration scale, including cooperativity factors that reflect the ratio of binding constants. (Similarly, proton linkage analysis in gITC can be carried out with unknown incompetent fractions of binding partners [24]. ) These can be extracted from gITC analysis if, for each component, the concentration errors can be constrained to be identical for all experiments.…”

“…Beyond merely organizing separate complementary experiments into a hierarchical, multi-stage interpretation, global analysis takes full advantage of all possible constraints of the model and the full statistics of the data by simultaneously and directly fitting all experimental data with one explicit global model. Natural applications of gITC analysis are multi-site binding processes of homo- and hetero-oligomerizing macromolecules [9–11,13,14,15 ( this volume ),23,33–37], displacement experiments [22,38,39], and protonation-linked and other linked binding analyses varying temperature to determine heat capacity changes or buffer composition to determine salt or other co-factor linkage [24,26,28]. …”

“…SEDPHAT is a computational platform for global analysis of data from various biophysical techniques, including gITC, which has been widely adopted in ITC applications [11–14,15 ( this volume ),17,19,24,33–62]. Distinguishing aspects of SEDPHAT in comparison with other gITC platforms include the absence of data-specific or model-specific programming by the user, allowing for seamless combination of titration experiments in a graphical user interface, and offering many different pre-programmed multi-site models for two- and three-component systems [9].…”

Fitting two- and three-site binding models to isothermal titration calorimetric data

“…Thus, the error surface around the best solution is systematically sampled and evaluated. This one-dimensional view of the surface is called an “error surface projection” [35,39]. No special computations are required of the user to utilize this strategy; it is fully implemented in this version of SEDPHAT, and it usually only takes seconds to report an error interval for globally analyzed ITC data.…”

Heat of adsorption of surfactants and its role on nanoparticle stabilization

“…Isothermal titration calorimetry (ITC) is a powerful technique for characterizing polymernanoparticle interaction [ 9 ], surfactant-polymer 10 or 11 polymeric particle interactions [10][11][12], micelle formation [ 13,14], and bimolecular interactions [15]. This method is based on the repetitive titration of a certain amount of ligands (e.g.…”