Membrane protein interaction studies using microscale thermophoresis

A common challenge in biophysical studies of membrane proteins is the choice of an adequate model membrane or membrane mimetic. Commonly used mimetics (detergents, liposomes) often suffer from well known limitations (adverse modification of protein structure, inhomogeneous protein distribution, etc.), thus prohibiting extensive studies on membrane proteins. However, the recently developed ‘nanodisc’ membrane mimetic sytem has helped alleviate some of these shortcomings. Nanodiscs are self assembled proteolipid particles, wherein two copies of an apolipoprotein A-I derived recombinant membrane scaffold protein (MSP) clasp a lipid patch, and seal the hydrophobic edge of the bilayer from water. The incorporation of a membrane protein into the nanodiscs is accomplished by its addition to the initial lipid and MSP mixture. Microscale thermophoresis (MST) is a recently developed technology that studies interactions based on the differential movement of biomolecules and their complexes in a microscopic temperature gradient. MST is highly sensitive, since it depends on changes in the size, charge and hydration shell of molecules. It measures interactions in free solution (no coupling required) and needs only very low sample volumes. It thus provides advantages over existing techniques like fluorescence correlation spectroscopy (FCS), isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). The use of nanodiscs in MST-based interaction studies of membrane proteins with ligands, or of lipid membranes with soluble peptides is very appealing. We thus explored this approach experimentally. Potential pitfalls of the method (aggregation of nanodiscs, undesired interactions with the capillary walls, etc.) were addressed and ways to overcome such difficulties are presented. Finally, MST was successfully applied for the measurement of binding affinities between various membrane proteins and their ligands. - See more at: http://bps2013.conferencespot.org/2862-Pos/#sthash.GzWWnFZK.dpu