Measuring the conformational stability of membrane proteins using the UNit

Introduction Use of extrinsic dyes to study protein unfolding Measurement of thermal stability is a valuable tool for assessing the suitability of proteins to a wide range of applications, including their use as therapeutic drugs or food additives. Indeed, researchers commonly require practical methods to rapidly screen conditions that afford the best environment for a particular protein. The UNit provides a route to obtaining such thermal stability information in a high throughput manner by probing intrinsic fluorescence and static light scattering changes of up to 48 samples simultaneously. In certain instances, it is helpful to expand the repertoire of optical probes used to assess the stability of proteins, such as by adding fluorescent dyes (e.g. 1-Anilinonaphthalene-8-Sulfonic Acid (ANS), SYPRO orange) that are sensitive to the amount of exposed hydrophobic residues. In other cases, the user can monitor the status of additional sensors such as the fluorophore present in green fluorescent protein (GFP). The UNit with an optional 375 nm laser has been designed to measure the thermal stability of both globular and integral membrane proteins using an extrinsic dye that covalently and specifically interacts with the unfolded form of the proteins. This method should be of particular importance for gaining stability information for membrane proteins.