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

Abstract

As isothermal titration calorimetry (ITC) gains popularity for the characterization of enthalpies and equilibrium association constants of simple 1:1 biomolecular interactions, its use for more complex systems is growing. The method is increasingly used to study interactions in which a single binding partner (molecule “A”) interacts with multiple copies of a second partner (“B”); thus examinations of ABB and ABBB interactions are not uncommon. The structure of ITC data (commonly formatted as isotherms) has a strong bearing on the ability of the researcher to extract the necessary parameters from them. Usually, only 10-30 injections are recorded in a single ITC experiment. Even if replicates are performed, the data must support the extraction of up to twelve parameters from an ABBB system conducted in triplicate. Further, the refinement of some of the parameters is largely driven by only a subset of the data. The ability of ITC data to guide the deterministic estimation of these parameters may therefore be questioned. This work assesses the ability of both empirical and simulated ITC data of ABB and ABBB systems to support the simultaneous estimation of the desired thermodynamic parameters. The results demonstrate that multiphasic isotherms tend to (but do not always) support the estimation of multiple parameters. On the other hand, uniphasic data obtained from multi-site binding systems are more problematic. In all cases, a thorough exploration of how precisely the estimated parameters are specified by the data is justified.

On the acquisition and analysis of microscale thermophoresis data

“…It is also quicker. The variance-covariance interval is usually quite small and is based on assumptions of local error-surface behavior that may not be true, while those derived from the Monte Carlo and bootstrap methods are slower and can neglect parameter correlations [49]. Thus, the final, reported K D for the HIF-E/ARNT-R* interaction was 820 [670, 990] nM.…”

Dissecting ITC data of metal ions binding to ligands and proteins

“…Protocols have been established in the literature regarding proper fitting procedures and error analysis [30]. New models describing more complex ligand/biomolecule interactions have been developed and global methods of analysis used to further substantiate fit parameters are being implemented [31][32][33][34].…”

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

“…Unfortunately, similar to most binding saturation curves, individual titration isotherms usually have relatively low intrinsic information content, due to the shallow concentration dependency of complex formation predicted by mass action law [15 ( this volume )]. Furthermore, dependent on the interaction enthalpies, even for multi-site systems only a single transition may be observed.…”

“…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].…”