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Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry
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Complementary Experimental Methods to Obtain Thermodynamic Parameters of Protein Ligand Systems
Shilpa Mohanakumar1, Namkyu Lee1, Simone Wiegand1,2
1IBI-4-Biomacromolecular Systems and Processes, Forschungszentrum Jülich GmbH, D-52428 Jülich, Germany.
International Journal of Molecular Sciences
|November 26, 2022
Summary
Thermophoresis quantifies biomolecular interactions by relating non-equilibrium Soret coefficients to equilibrium thermodynamic properties. This study validates the approach using chemical and protein binding systems, showing good agreement between methods.
Area of Science:
- Physical Chemistry
- Biophysics
- Chemical Thermodynamics
Background:
- Thermophoresis is a promising technique for studying biomolecular interactions, but its underlying physical mechanisms remain unclear.
- Attribution to hydration layer changes upon binding is common, yet a direct link to equilibrium properties is sought.
- Non-equilibrium thermophoretic coefficients are investigated for their relation to equilibrium thermodynamic properties.
Purpose of the Study:
- To investigate the relationship between non-equilibrium thermophoretic coefficients and equilibrium thermodynamic properties.
- To validate thermophoresis as a quantitative tool for biomolecular interactions by comparing it with isothermal titration calorimetry.
- To assess the influence of fluorescent labeling on binding behavior for microfluidic measurements.
Main Methods:
- Thermal Diffusion Forced Rayleigh Scattering (TDFRS) for non-equilibrium thermophoretic measurements.
- Isothermal Titration Calorimetry (ITC) for equilibrium thermodynamic measurements.
- Utilizing Eastman's expression to relate Soret coefficient (ST) to Gibb's free energy (ΔG).
Main Results:
- The Gibb's free energies calculated from thermophoretic data (ST) showed agreement with ΔG obtained from ITC for all tested systems.
- The chelation of EDTA with CaCl2 and the binding of Bovine Carbonic Anhydrase I (BCA I) to 4-fluorobenzenesulfonamide (4FBS) and pentafluorobenzenesulfonamide (PFBS) were successfully analyzed.
- Fluorescent labeling significantly influenced the binding behavior of the protein-ligand system, as revealed by ITC re-examination.
Conclusions:
- Thermophoresis, quantified by the Soret coefficient, can accurately predict equilibrium thermodynamic properties like Gibb's free energy.
- The study provides a foundation for using thermophoresis as a reliable method for biomolecular interaction analysis.
- Careful consideration of labeling effects is crucial when employing fluorescent techniques for biomolecular interaction studies.
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