Phospholipid-Interacting Proteins by Solution-State NMR Spectroscopy

Signaling lipids are found in specific subcellular membranes, where they recruit and regulate cytosolic proteins and contribute to bilayer structure and dynamics. These interactions are vital for signaling and membrane trafficking pathways and contribute to the organization, growth, and differentiation of the cell. However, the analysis of the physical and chemical mechanisms of membrane interaction and lipid recognition is technically challenging, motivating the development of new NMR methods to study lipid and bilayer binding by peripheral membrane proteins in solution. We describe methods that have been optimized for the FYVE and phox (PX) domains of the EEA1 and Vam7p proteins, respectively, both of which specifically recognize phosphatidylinositol 3-phosphate (PtdIns3P ) within endocytic membranes. Solution-state NMR methods were used to characterize the phosphoinositide and membrane interaction sites and affinities and can be used to illustrate protein:micelle structures and phospholipid specificities. The methods are generally applicable and can be used to discover and characterize the phospholipid interactions of other membrane-interacting protein domains.