Chemical and Materials Sciences: Research Findings Vol. 7

Microscale thermophoresis (MST) is a powerful immobilisation-free biophysical technique that is very useful for measuring biomolecular interactions with membrane proteins. MST is based on the physical phenomenon that particles move within temperature gradients, which is affected by their size, charge, hydration shell and conformation. This study aims to present an updated overview and practical application of MST as a biophysical technique for characterising membrane protein interactions. A fluorescent target molecule must be included in the sample to observe the movement of particles by MST. The intrinsic aromatic residues of membrane proteins can be used as the fluorophore for MST (label-free MST), or membrane proteins can be labelled with a range of fluorescent dyes or conjugated with fluorescent proteins (labelled MST). In the MST experiment, the membrane protein can be titrated with an unlabelled binding partner (e.g. other proteins, peptides, small molecules, ions) for quantifying interactions. MST is highly sensitive, using relatively small amounts of sample, and it has no limitations on the size of the target biomolecule, on the affinity of the interaction or on the composition of the buffer and other sample components. Membrane proteins can be characterised by MST in cell lysates, native membranes, solubilised in detergents, and reconstituted in lipids or other membrane mimetics. Different types of membrane proteins have had biomolecular interactions characterised by MST, including SARS-CoV-2 spike protein, GPCRs and other receptors, sensor kinases, ion channels, aquaporins and transport proteins. MST has great future potential in the drug discovery pipeline for medium- to high-throughput compound screening with membrane protein drug targets. This will be accelerated by technical developments in MST that enhance throughput, increase sensitivity and minimise interference.