Complementary experimental methods to obtain thermodynamic parameters of protein ligand systems

TL;DR

This study compares non-equilibrium thermophoresis data with equilibrium calorimetry data to relate thermophoretic behavior to thermodynamic parameters in protein-ligand systems, revealing consistent free energy estimates.

Contribution

It demonstrates that thermophoretic measurements can be related to thermodynamic parameters, providing a new approach to study biomolecular interactions.

Findings

Thermophoretic data's Gibbs free energy aligns with calorimetric data.

Fluorescent labeling affects binding behavior significantly.

Thermophoresis can be used to estimate thermodynamic parameters.

Abstract

In recent years, thermophoresis has emerged as a promising tool for quantifying biomolecular interactions. The underlying physical effect is still not understood. To gain deeper insight, we investigate whether non-equilibrium coefficients can be related to equilibrium properties. Therefore, we compare thermophoretic data measured by thermal diffusion forced Rayleigh scattering (TDFRS) (which is a non-equilibrium process) with thermodynamic data obtained by isothermal titration calorimetry (ITC) (which is an equilibrium process). As a reference system, we studied chelation reaction between ethylenediaminetetraacetic acid (EDTA) and calcium chloride (CaCl$_2$) to relate the thermophoretic behavior quantified by the Soret coefficient $S_{\mathrm T}$ to the Gibb's free energy $\Delta G$ determined in the ITC experiment using an expression proposed by Eastman [J. Am. Chem. Soc. 50, 283 (1928)]. Finally, we have studied the binding of the protein Bovine Carbonic Anhydrase I (BCA I) to two different benzenesulfonamide derivatives: 4-fluorobenzenesulfonamide (4FBS) and pentafluorobenzenesulfonamide (PFBS). For all three systems, we find that the Gibb' free energies calculated from $S_{\mathrm T}$ agree with $\Delta G$ from the ITC experiment. In addition, we also investigate the influence of fluorescent labeling, which allows measurements in a thermophoretic microfluidic cell. Re-examination of the fluorescently labeled system using ITC showed a strong influence of the dye on the binding behavior.