Journal
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
Volume 23, Issue 22, Pages -Publisher
MDPI
DOI: 10.3390/ijms232214198
Keywords
thermophoresis; thermodiffusion; Soret effect; protein-ligand binding; hydration effects; entropy-enthalpy compensation; thermal diffusion forced Rayleigh scattering; isothermal titration calorimetry; thermophoretic microfluidic cell
Funding
- International Helmholtz Research School of Bio-physics and Soft Matter (BioSoft)
- Humboldt foundation
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In this study, we compared data obtained from the non-equilibrium process of thermal diffusion forced Rayleigh scattering (TDFRS) with data obtained from the equilibrium process of isothermal titration calorimetry (ITC). The results showed that the thermophoretic behavior quantified by the Soret coefficient S_T agreed with the Gibb's free energy Delta G determined in the ITC experiment. We also investigated the influence of fluorescent labeling on the binding behavior and found that the dye had a significant impact.
In recent years, thermophoresis has emerged as a promising tool for quantifying biomolecular interactions. The underlying microscopic physical effect is still not understood, but often attributed to changes in the hydration layer once the binding occurs. 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 the chelation reaction between ethylenediaminetetraacetic acid (EDTA) and calcium chloride (CaCl2) to relate the thermophoretic behavior quantified by the Soret coefficient S-T to the Gibb's free energy Delta G determined in the ITC experiment using an expression proposed by Eastman. 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's free energies calculated from S-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.
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