Universität zu Köln

Mohanakumar, Shilpa ORCID: 0000-0001-7098-0065, Lee, Namkyu ORCID: 0000-0002-0342-9764 and Wiegand, Simone ORCID: 0000-0001-6333-1956 (2022). Complementary Experimental Methods to Obtain Thermodynamic Parameters of Protein Ligand Systems. Int. J. Mol. Sci., 23 (22). BASEL: MDPI. ISSN 1422-0067

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Abstract

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.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Mohanakumar, Shilpa UNSPECIFIED orcid.org/0000-0001-7098-0065 UNSPECIFIED Lee, Namkyu UNSPECIFIED orcid.org/0000-0002-0342-9764 UNSPECIFIED Wiegand, Simone UNSPECIFIED orcid.org/0000-0001-6333-1956 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-671231
DOI:	10.3390/ijms232214198
Journal or Publication Title:	Int. J. Mol. Sci.
Volume:	23
Number:	22
Date:	2022
Publisher:	MDPI
Place of Publication:	BASEL
ISSN:	1422-0067
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language CARBONIC-ANHYDRASE INHIBITORS; ISOTHERMAL TITRATION CALORIMETRY; THERMAL-DIFFUSION; SORET COEFFICIENT; CONFORMATIONAL ENTROPY; TEMPERATURE-DEPENDENCE; SOLVENT INTERACTIONS; CALCIUM-EDTA; BINDING; COMPLEXES Multiple languages Biochemistry & Molecular Biology; Chemistry, Multidisciplinary Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/67123