Cell-sized droplet interfaces reorganize protein secondary structures through confinement-enhanced membrane interactions

Abstract

Cell membranes can regulate protein organization, yet it remains unclear whether membrane-associated structural alterations primarily reflect membrane-induced destabilization or the reorganization of proteins that are already destabilized. How such membrane effects are amplified under cellular-scale confinement also remains poorly understood. Here, we investigate these questions using cell-sized lipid-coated droplets, where the high surface-area-to-volume ratio enhances the contribution of membrane interfaces. Native serum albumin and lysozyme showed little structural reorganization, whereas their thermally denatured forms exhibited pronounced $\beta$-sheet formation within small droplets when membrane interactions were attractive. For denatured albumin, $\beta$-sheet-rich organization increased progressively with protein--membrane attraction, while denatured lysozyme selectively formed a localized $\beta$-sheet-rich shell at a complementary anionic membrane. Circular dichroism spectroscopy independently supported the confinement-enhanced increase in $\beta$-sheet content of denatured albumin. Fluorescence recovery measurements further revealed strong interfacial arrest in both systems. Together, these results show that membrane interactions can promote structural reorganization of already destabilized proteins through electrostatic recruitment, while cell-sized confinement strongly amplifies the resulting membrane-dependent structural response. Our findings establish cell-sized droplet interfaces as a materials platform for spatially controlling protein structural organization through the interplay of interfacial chemistry and confinement.