Many membrane-associated cytosolic proteins contain unstructured N-terminal domains that transition into membrane solvated amphipathic helices upon binding. Despite their recognized roles in organelle recognition, these inducible amphipathic helices remain poorly understood due to the innate experimental complexity of isolating and quantifying membrane binding of these domains without their host proteins. To address this, we have developed a recombinant protein platform that solubilizes inducible amphipathic helices with a fluorescently tagged SUMO (Small Ubiquitin-like Modifier) protein and quantifies membrane binding to lipid vesicles via fluorescence anisotropy. Despite the larger apparent molecular-weight of the protein-vesicle system, we detect a considerable decrease in fluorescence anisotropy, signaling an increase in the fluorophore’s rotational diffusion. To explore this molecular mechanism, we employ time-resolved spectroscopic techniques and probe the dynamics of the fluorophore in the nanosecond regime. Combining these high-precision techniques with targeted mutations within the SUMO protein, we extract structural information that suggests the decrease in fluorescence anisotropy occurs via a regional “unfolding” in the domain of the fluorophore. Leveraging this molecular mechanism allows for high-throughput protein-vesicle binding measurements, enabling comprehensive parameter searches for the biochemical properties driving subcellular protein localization.
Margaritakis et al. (Sun,) studied this question.