Bacterial-Mimetic Membranes Reveal Hopanoid-Associated Domain-Like Lateral Heterogeneity

Bacterial membranes are increasingly recognized as laterally heterogeneous assemblies whose local organization can regulate transport, stress adaptation, and interactions with membrane-active compounds. However, the physical principles by which bacterial lipid composition generates spatially distinct membrane environments remain poorly understood, partly because experimentally tractable models that reproducibly exhibit lateral segregation and permit quantitative characterization are limited. Here, we show that a multicomponent, hopanoid-containing bacterial-inspired membrane spontaneously self-organizes into laterally distinct physical environments that are consistently resolved in experiments and molecular simulations. Giant unilamellar vesicles prepared from a lipid mixture inspired by lipid classes found in hopanoid-producing bacteria, including Methylorubrum extorquens, exhibit stable micron-scale lateral segregation, which is independently reproduced in compositionally matched coarse-grained molecular dynamics simulations. The resulting membrane environments differ in composition, packing, order, mobility, and mechanical properties. Hopanoid-associated domain-like regions are more condensed, more ordered, and less mobile, whereas PE-rich non-domain regions remain more laterally expanded and exhibit faster lipid diffusion. At the global level, the bacterial-inspired membrane displays greater bending rigidity than a fluid phosphatidylcholine reference, while ATR-FTIR measurements independently support increased lipid-chain ordering and denser membrane packing. Together, these findings establish that hopanoid-containing bacterial-inspired membranes can spontaneously generate coexisting physical environments whose distinct local properties collectively determine global membrane behavior. The resulting experimental-computational framework provides a quantitative basis for investigating bacterial-like membrane heterogeneity and how local composition, dynamics, and mechanics govern responses to membrane-targeting perturbations.