Measuring the Interaction of Sterols and Steroids with Proteins by Microscale Thermophoresis.

Abstract

Determining the affinity and specificity of protein-lipid interactions is crucial for understanding the physiological function and mode of action of signaling lipids, including steroids. Here we describe a method that relies on microscale thermophoresis (MST) to monitor the binding of sterols and steroids to proteins. The protein of interest is expressed as a polyhistidine-tagged fusion in E. coli and purified by affinity chromatography on a nickel-based resin. The purified protein is then labeled with a fluorescent dye and incubated with a serial dilution of the lipid ligand. The protein-ligand interaction is monitored by MST, which detects the fraction of the protein bound to the ligand based on its altered mobility in a thermal gradient. A binding curve fitted to the measured data points is then used to calculate the corresponding dissociation constant.