Rotational conformation change of protein upon binding to a vesicle yields lower steady state fluorescence anisotropy values.

Peripheral membrane-binding proteins often contain a domain that transitions from a disordered state to an amphipathic helix upon binding to liposomes. To facilitate investigating the properties of such peptides, we introduce a novel, generalizable platform able to probe and characterize the liposome-peptide interaction through fluorescence anisotropy in a high-throughput manner. Surprisingly, instead of the anticipated increase in fluorescence anisotropy upon binding, we detect a significant decrease (indicating a higher rotational diffusion of the bound state). This unexpected decrease is directly correlated to the binding interaction as confirmed by rigorous tryptophan fluorescence measurements. To further understand this mechanism, we characterize the nature of the interaction using time-based spectroscopic techniques, revealing a key conformational change in the protein’s structure and the local mobility of the fluorophore. This dynamic shift, when sampled in a time-averaged manner overwhelms the slower rotation of the protein-liposome system and is translated into the measured steady state anisotropy decrease. Our platform exploits this interaction and provides a high-throughput screening tool able to determine the specific regions in membrane phase space where protein binding is maximized and paves the way towards uncovering the peptide code responsible for precise protein subcellular localization.