Thermophoresis: The Case of Streptavidin and Biotin

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.

Numerical Analysis of Thermophoresis of a Charged Spheroidal Colloid in Aqueous Media

“…The thermophoresis approach has been used to separate, trap, and concentrate nanoparticles in liquid media [ 4 , 5 , 6 , 7 ]. For biological and biocompatible compounds [ 8 ] in aqueous solutions, the thermophoretic techniques provide useful tools to quantify the protein−ligand binding constants [ 9 ] and the copy numbers of noncoding RNA [ 10 ], examine the hydration layer [ 11 ], and determine the biomolecular interactions [ 12 ].…”

Thermophoretic Micron-Scale Devices: Practical Approach and Review

“…Because the most important application of thermophoresis is the investigation of protein-ligand binding, which generally have to be stabilized in a buffer solution, it is desirable to have an optical transparent cell, which can be operated under a fluorescence microscope. In this way, the distribution of the proteins can be determined using a fluorescent label without the buffer components contributing to the signal, as is the case with optical methods that are based on refractive index contrast [ 33 ]. To get as close as possible to the ideal case of a one-dimensional temperature profile, a channel (cf.…”

“…This led to the development of new methods that require small sample volume supporting the limited availability of biological samples of interest, such as proteins and ligands. There are two main application routes: on one hand, thermophoresis is used in combination with convective flow to accumulate and replicate biomolecules [ 23 , 24 , 25 , 26 , 27 ], and on the other hand, thermophoresis is applied as analytical tool to monitor binding reactions of biomolecules, such as between proteins and ligands [ 28 , 29 , 30 , 31 , 32 , 33 ]. The working horse in this field is the commercially available microscale thermophoresis (MST), which is used to determine the dissociation constant , giving access to the change in Gibbs free energy.…”

“…This also shines some light on disease’s mechanism in medical applications such as influenza [ 34 ], Alzheimer [ 35 ] and corona [ 36 ]. It would be desirable to develop methods that provide quantitative thermophoretic values, which can be used to develop theories to explain conformational transitions and hydration layer changes during the protein-ligand binding process in order to obtain a more fundamental understanding [ 32 , 33 ]. A deeper understanding from the theoretical side will certainly help to optimize the search for suitable pharmaceutical compounds.…”

Temperature profile characterization with fluorescence lifetime imaging microscopy in a thermophoretic chip

“…However, while general trends of microscale devices are often confirmed by other experiments [ 13 , 15 , 20 , 22 ], a direct comparison of results of the same system with validated methods as done for low-molecular weight compounds remains challenging [ 23 ]. For instance, it turned out that thermophoretic measurements of a protein–ligand system with an established method are difficult to analyze as it is not always possible to separate signal contributions stemming from buffer, ligand and protein simultaneously [ 24 ]. Those difficulties could certainly be avoided, if the thermophoretic motion of a protein could be monitored directly using a thermophoretic chip.…”