MicroScale Thermophoresis: Interaction analysis and beyond

Moran Jerabek-Willemsen¹, Timon André^1,2, Randy Wanner^1,3, Heide Marie Roth¹, Stefan Duhr¹, Philipp Baaske¹, Dennis Breitsprecher^1§

¹Nanotemper Technologies GmbH Flößergasse 4, 81369 Munich, Germany
²Heidelberg University Biochemistry Center (BZH) Im Neuenheimer Feld 328 D-69120 Heidelberg

³Department of Pharmacy Pharmaceutical Technology & BiopharmaceuticsButenandtstr. 5, D-81377 München

MicroScale thermophoresis (MST) is an exceptionally powerful technique to quantify biomolecular interactions. It is based on thermophoresis, the directed movement of molecules in a temperature gradient, which strongly depends on a variety of molecular properties such as size, charge, hydration shell or conformation. Thus, this technique is highly sensitive to virtually any change in molecular properties, allowing for a precise quantification of molecular events independent of the size or nature of the investigated specimen.

During an MST experiment, a temperature gradient is induced by an infrared laser. The directed movement of molecules through the temperature gradient is detected and quantified using either covalently attached or intrinsic fluorophores. By combining the precision of fluorescence detection with the variability and sensitivity of thermophoresis, MST provides a flexible, robust and fast way to dissect molecular interactions.

In this work, we present recent progress and developments in MST technology and focus on MST applications beyond standard biomolecular interaction studies. By using different model systems, we introduce alternative MST applications – such as determination of binding stoichiometries and binding modes, analysis of protein unfolding, thermodynamics and enzyme kinetics – and also demonstrate the capability of MST to quantify high-affinity interactions with K_ds in the low pM range as well as protein-protein interactions in pure cell lysates.