In vitro methods for studying protein-lipid droplet interactions

Lipid droplets (LDs) are major organelles that store cellular energy in the form of a neutral lipid core surrounded by a phospholipid monolayer. Proteins involved in the biogenesis and regulation of LDs associate with them via distinct mechanisms: they insert into the ER membrane via a hydrophobic hairpin and diffuse to the LD monolayer, interact with LD-resident proteins, or directly bind to the LD monolayer via amphipathic helices (AHs) that are highly sensitive to lipid packing. This chapter presents optimized methods to study the binding of AHs or recombinant proteins to fluorescent artificial lipid droplets (aLDs) or LD-mimicking liposomes whose membranes have lipid packing defects. We first provide a practical and comprehensive guide to the production and characterization of aLDs and liposomes and to the design and generation of amphipathic peptides and recombinant proteins. Second, we present complementary in vitro approaches, including flotation, pull-down, and aggregation assays, to measure the interaction of peptides or proteins with aLDs and liposomes, explaining how to execute and analyze these binding assays.