Cell-Sized Droplet Interfaces Reorganize Protein Secondary Structures through Confinement-Enhanced Membrane Interactions

Cell membranes are recognized as regulators of protein organization, yet it remains unclear whether membrane-associated structural transitions arise from membrane-induced destabilization or from the reorganization of proteins already destabilized before membrane contact. Here, we address this question using cell-sized lipid-coated droplets. Native serum albumin and lysozyme showed little structural reorganization, whereas their thermally denatured forms underwent membrane-dependent β -sheet formation. Denatured albumin exhibited progressively enhanced β -sheet-rich organization with increasing protein–membrane attraction, whereas denatured lysozyme selectively formed a localized β -sheet-rich shell at a complementary anionic membrane. Fluorescence recovery measurements revealed strong interfacial arrest in both systems. Together, these results show that membrane interfaces reorganize already destabilized proteins through electrostatic recruitment and that confinement amplifies this membrane effect. Our findings establish cell-sized droplet interfaces as active regulators of protein structural organization and provide a physical framework linking membrane confinement to amyloid-like structural transitions.