Determining the Relative Affinity of ORPs for Lipid Ligands Using Fluorescence and Thermal Shift Assays

Lipid transfer proteins (LTPs) are specialized proteins that convey specific lipids across the cytosol to regulate the lipid composition of organelles and the plasma membrane. Quantifying to which extent these LTPs recognize and transfer various lipid species and subspecies is of prime interest to define their cellular role(s). Here, we describe how to measure in vitro the relative affinity of Osh6p, a yeast phosphatidylserine (PS)/phosphatidylinositol 4-phosphate (PI(4)P) exchanger belonging to the oxysterol-binding protein(OSBP)-related protein (ORP) family, for PS and phosphoinositide subspecies. First, we detail how to produce and purify Osh6p with high purity. Secondly, we describe how to measure its ability to bind PS, PI(4)P, and PI(4,5)P₂ by FRET-based and thermal shift assays using liposomes of defined composition. These protocols can allow further analysis of other ORPs or inspire the design of assays to characterize other LTPs. Notably, they can be helpful in defining how LTPs transfer phospholipids subspecies as a function of their acyl chains' length and unsaturation degree and, therefore, whether they can contribute to regulating the acyl chain composition of cell membranes.