Biomolecular Interaction Determination and Quantification by Microscale Thermophoresis

Abstract

MicroScale Thermophoresis (MST), an immobilization-free technology, is used to rapidly quantify biomolecular interactions (pM-mM). MST, the directed movement of molecules in optically generated microscopic temperature gradients, is monitored by fluorescence. Thermophoretic movement is highly sensitive to binding reactions, which affect the size, charge, conformation, and/or hydration shell of target molecules. Here, we demonstrate that MST can be used to quantify interactions between biomolecules of interest, including proteins, nucleic acids, ions, and more. In one example, the binding site of a protein-protein interaction is investigated via protein engineering. As MST is suitable for probe interactions with membrane proteins in detergent or liposomes, we also demonstrate the quantification of interactions with GPCRs in a label free format using tryptophan fluorescence. Thus, MST is a critical addition to the biophysical toolbox allowing greater user-flexibility for analyzing molecular interactions.