A proximity-based labelling approach to analyse lipid binding in proteins

Abstract

Membranes act as sites of several biochemical processes occurring inside a cell. Soluble proteins that bind these membranes often do so by interacting with resident lipids. A preliminary step in understanding these proteins is thus studying their binding specificity and affinity to these lipids. Various methods exist to probe for these interactions, often relying on liposomes as the membrane substrate of choice. Yet, distinguishing protein-lipid binding from protein-membrane binding is impossible on these substrates. In addition, liposomes suffer from several limitations, such as the involved preparation methodologies, longer-term structural instability and the amount of lipids consumed in each prep. Here, we propose detergent micelles as a simpler yet useful substrate for detecting and quantifying protein-lipid interactions. Detergents are doped with small amounts of test lipids along with a bifunctional photoactivable fluorescent lipid to form the substrate for protein binding. Proteins that bind the test lipid are covalently labelled by the bifunctional lipid in a proximity-based manner upon exposure to UV, which can then be detected and quantified by in-gel fluorescence as a readout for binding. At low concentrations of test lipid, this assay functions as a lipid-protein binding assay. The ease of preparation, along with the relatively inexpensive reagents and equipment required, make this a more accessible assay.