Abstract
Biophysical techniques such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) are routinely used to ascertain the global binding mechanisms of protein-protein or protein-ligand interaction. Recently, Dumas etal, have explicitly modelled the instrument response of the ligand dilution and analysed the ITC thermogram to obtain kinetic rate constants. Adopting a similar approach, we have integrated the dynamic instrument response with the binding mechanism to simulate the ITC profiles of equivalent and independent binding sites, equivalent and sequential binding sites and aggregating systems. The results were benchmarked against the standard commercial software Origin-ITC. Further, the experimental ITC chromatograms of 2′-CMP + RNASE and BH3I-1 + hBCLXL interactions were analysed and shown to be comparable with that of the conventional analysis. Dynamic approach was applied to simulate the SPR profiles of a two-state model, and could reproduce the experimental profile accurately.
Highlights
Biophysical techniques such as isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR) are used to thermodynamically and kinetically characterize the binding mechanism of the protein-ligand or protein-protein interactions, respectively [1,2,3,4]
Standard experimental data belonging to four different mechanisms such as (1) single site (RNAHH) (2) two sequential sites (PROTDB) (3) four sequential sites (PERSSON) and (4) two independent sites (FEOTF54) as provided in Origin-ITC software package were subjected to the conventional normalized delta heat’ data (NDH) based analysis [9,10]
The NDH data resulted from the integration of the simulated thermograms were identical to that of the NDH values obtained independently from conventional algebraic approach using the same set of fit parameters SI (1.7)
Summary
Biophysical techniques such as isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR) are used to thermodynamically and kinetically characterize the binding mechanism of the protein-ligand or protein-protein interactions, respectively [1,2,3,4]. ITC measures the heat released or absorbed during the protein-ligand interactions [1,2,3,5,6], whereas, SPR measures the change in reflective angle of the incident light caused by surface waves called ‘plasmon polaritons’. In ITC experiments, the thermogram with asymmetric gaussian-like peaks are numerically integrated and normalized with respect to the titrated ligand to obtain ‘normalized delta heat’ data (NDH) [9,10]. NDH data is analysed to obtain the stoichiometry, binding equilibrium, and enthalpy constants. In this article, we have incorporated both the instrument response and the binding mechanism within an integrated kinetic framework
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