Abstract
Thermophoretic behavior of a free protein changes upon ligand binding and gives access to information on the binding constants. The Soret effect has also been proven to be a promising tool to gain information on the hydration layer, as the temperature dependence of the thermodiffusion behavior is sensitive to solute–solvent interactions. In this work, we perform systematic thermophoretic measurements of the protein streptavidin (STV) and of the complex STV with biotin (B) using thermal diffusion forced Rayleigh scattering (TDFRS). Our experiments show that the temperature sensitivity of the Soret coefficient is reduced for the complex compared to the free protein. We discuss our data in comparison with recent quasi-elastic neutron scattering (QENS) measurements. As the QENS measurement has been performed in heavy water, we perform additional measurements in water/heavy water mixtures. Finally, we also elucidate the challenges arising from the quantiative thermophoretic study of complex multicomponent systems such as protein solutions.
Highlights
In recent years, thermophoresis, which is the mass transport caused by a temperature gradient, has gained a lot of interest [1]
Neutron scattering experiments probe the internal dynamics of the protein and can be used to determine the entropic change of the protein in ligand-binding reactions or in protein folding [30,31], while thermophoresis is more sensitive to the hydration layer and the accompanying entropic changes of the surrounding water molecules
Thermodiffusion of STV was measured by infrared thermal diffusion forced Rayleigh scattering (IR-TDFRS) [33,34]
Summary
In recent years, thermophoresis, which is the mass transport caused by a temperature gradient, has gained a lot of interest [1]. For lower log P-values, a stronger change of the Soret coefficient with temperature is observed This might indicate that a larger number of water molecules connect to the solute molecule via hydrogen bonds. Neutron scattering experiments probe the internal dynamics of the protein and can be used to determine the entropic change of the protein in ligand-binding reactions or in protein folding [30,31], while thermophoresis is more sensitive to the hydration layer and the accompanying entropic changes of the surrounding water molecules. D2 O [32] agree within experimental errors with calculated scattering curves based on high resolution crystal structures of free STV and its ligand-bound state (STV + B) This observation does not exclude differences in the interactions of the protein with H2 O and D2 O, respectively
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