Microscale Thermophoresis Analysis of Membrane Proteins
Nighat Nawaz
School of Biomedical Sciences (Astbury Building), University of Leeds, Leeds LS2 9JT, UK and Department of Chemistry, Islamia College Peshawar, Peshawar 25120, Pakistan.
Roshan Ali
Institute of Basic Medical Sciences, Khyber Medical University, Peshawar 25100, Pakistan.
Muhammad Ali
Institute of Basic Medical Sciences, Khyber Medical University, Peshawar 25100, Pakistan.
Iain W. Manfield
School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK.
Muhammad Kamran Taj
Centre for Advanced Studies in Vaccinology and Biotechnology, University of Balochistan, Quetta 08763, Pakistan.
Mohammad Zahid Mustafa
Centre for Advanced Studies in Vaccinology and Biotechnology, University of Balochistan, Quetta 08763, Pakistan.
Simon G. Patching *
School of Biomedical Sciences (Astbury Building), University of Leeds, Leeds LS2 9JT, UK.
*Author to whom correspondence should be addressed.
Abstract
Microscale thermophoresis (MST) is an analytical technique for measuring biomolecular interactions. It is based on the physical phenomenon that particles move within temperature gradients, which is affected by their size, charge, hydration shell and conformation. The MST sample must contain a fluorescent target molecule used to observe the movement of particles, and this can be titrated with an unlabelled binding partner for quantifying the interaction. 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. This makes MST ideally suited to characterising interactions with membrane proteins, which can be studied in cell lysates, native membranes, solubilised in detergents or reconstituted in lipids. The intrinsic aromatic residues of membrane proteins have been used as the fluorophore for MST (label-free MST) or membrane proteins have been labelled with a range of fluorescent dyes or conjugated with fluorescent proteins (labelled MST). The different types of membrane proteins that have had biomolecular interactions characterised by MST include the SARS-CoV-2 spike protein, GPCRs and other receptors, sensor kinases, ion channels, aquaporins, and transport proteins.
Keywords: Biomolecular interactions, fluorescent labelling, ligand binding, drug screening, SARS-CoV-2 spike protein, GPCRs, receptors, ion channels, aquaporins, transport proteins