Recombinant protein platform for high-throughput investigation of peptide-liposome interactions via fluorescence anisotropy depolarization.

Many cytosolic proteins critical to membrane trafficking and function contain an unstructured domain that can bind to specific membranes, with a transition into an amphipathic helix induced upon membrane association. These inducible amphipathic helices often play a critical role in organelle recognition and subsequent function, but the tools used to characterize affinity towards specific membranes are low-throughput and dependent on the solubility of the inducible amphipathic helix. Here, we introduce a modular recombinant protein platform for rapidly measuring the binding affinity of inducible amphipathic helices towards a variety of membrane compositions and curvatures using high-throughput fluorescence anisotropy measurements. Inducible amphipathic helices are solubilized with a fluorescently-tagged small ubiquitin-like modifier (SUMO) protein and binding to membranes quantified by leveraging the unexpected decrease in fluorescence anisotropy upon binding, a phenomenon previously observed but not well understood. By using fluorescence anisotropy decay measurements, solution NMR experiments, and solution FRET, we deduce that this phenomenon likely occurs due to the local increase in fluorophore motion upon binding to the membrane enabled by vesicle membrane charge under low-salt conditions. This recombinant protein platform can be readily applied to any inducible amphipathic helix of interest, allowing for investigation of specific membrane biochemical parameters facilitating binding.