On the acquisition and analysis of microscale thermophoresis data

A comprehensive understanding of the molecular mechanisms underpinning cellular functions is dependent on a detailed characterization of the energetics of macromolecular binding, often quantified by the equilibrium dissociation constant, K_D. While many biophysical methods may be used to obtain K_D, the focus of this report is a relatively new method called microscale thermophoresis (MST). In an MST experiment, a capillary tube filled with a solution containing a dye-labeled solute is illuminated with an infrared laser, rapidly creating a temperature gradient. Molecules will migrate along this gradient, causing changes in the observed fluorescence. Because the net migration of the labeled molecules will depend on their liganded state, a binding curve as a function of ligand concentration can be constructed from MST data and analyzed to determine K_D. Herein, simulations demonstrate the limits of K_D that can be measured in current instrumentation. They also show that binding kinetics is a major concern in planning and executing MST experiments. Additionally, studies of two protein-protein interactions illustrate challenges encountered in acquiring and analyzing MST data. Combined, these approaches indicate a set of best practices for performing and analyzing MST experiments. Software for rigorous data analysis is also introduced.