Membrane (and Soluble) Protein Stability and Binding Measurements in the Lipid Cubic Phase Using Label-Free Differential Scanning Fluorimetry

Label-free differential scanning fluorimetry (DSF) is a relatively new method for evaluating the stability of proteins. It can be used as a screening tool for downstream applications such as crystallization. The method is attractive in that it requires miniscule quantities of proteins, it can be performed using intrinsic tryptophan and tyrosine fluorescence, and, with the right equipment, it is easy to perform. To date, the method has been used with proteins in liquid solutions and dispersions. It was of interest to determine if DSF could be used with membrane proteins in the lipid cubic phase (LCP), which increasingly is being used for crystallization in support of structure–function studies. The cubic phase is viscous. Furthermore, in coexistence with excess aqueous solution, as happens during crystallization trials, it can become turbid and scatter light. The concern was that these features may render the mesophase unsuitable for DSF analysis. However, using lysozyme and four integral membrane proteins...