Flotation Assay With Fluorescence Readout to Study Membrane Association of the Enteroviral Peripheral Membrane...

Enteroviruses are abundant pathogens of humans and animals. Their replication is strictly dependent on the conserved, viral AAA+ ATPase 2C. 2C is an oligomerizing, peripheral membrane protein, and its low solubility as recombinant protein has hampered functional studies of the full-length, recombinant protein bound to a membrane. Here, we describe a modification of the classical, ultracentrifugation-based liposome flotation assay optimized to study the interaction of recombinant 2C with membranes and the functions of membrane-bound, full-length recombinant 2C. The assay takes advantage of the high solubility of recombinant 2C while fused to a maltose-binding protein. Removing this solubility-enhancing tag by specific protease cleavage in the presence of liposomes allows 2C to associate with membranes prior to aggregating. Fluorophore labeling of protein and liposomes allows rapid and precise quantitation of 2C's association with membranes. This assay is adaptable to any peripheral membrane protein that can be fluorophore-labeled and expressed as a solubility-enhancing fusion protein. Key features • This protocol extends widely used liposome flotation assays to low-solubility peripheral membrane proteins, such as the enteroviral protein 2C. • 2C is expressed and purified as a fusion protein with a solubility-enhancing tag, which is cleaved off in the presence of liposomes. • Fluorophore-labeling of liposomes and protein facilitates quantitative readout of protein association with membranes. • The protein-conjugated liposomes can also be used for other studies using, e.g., dynamic light scattering, cryo-EM, and enzymatic activity assays. • The protein-conjugated liposomes can also be used for other studies using, e.g., dynamic light scattering, cryo-EM, and enzymatic activity assays.