A model of the outer membrane of Gram-negative bacteria was created by the deposition of a monolayer of purified rough mutant lipopolysaccharides at an air/water interface. The density profiles of monovalent (K þ ) and divalent (Ca 2þ ) cations normal to the lipopolysaccharides (LPS) monolayers were investigated using grazingincidence X-ray fluorescence. In the absence of Ca 2þ , a K þ concentration peak was found in the negatively charged LPS headgroup region. With the addition of CaCl 2 , Ca 2þ ions almost completely displaced K þ ions from the headgroup region. By integrating the experimentally reconstructed excess ion density profiles, we obtained an accurate measurement of the effective charge density of LPS monolayers. The experimental findings were compared to the results of Monte Carlo simulations based on a coarse-grained minimal model of LPS molecules and showed excellent agreement.monolayer | Monte Carlo simulation | electrostatics | biological interface B iological surfaces expose a variety of charged macromolecules that interact with various sorts of ions under physiological conditions. However, despite the crucial role of charged macromolecules in modulating the interaction between cells and their surrounding environments, the quantitative understanding of electrostatics at such soft, complex interfaces still remains a general scientific challenge. For example, the outer membrane surface of Gram-negative bacteria is mainly composed of lipopolysaccharides (LPSs) (1), whose negatively charged saccharide head groups stabilize the structural integrity of bacteria and protect bacteria against their environment. Several in vivo studies (2, 3) demonstrated that bacteria increase their resistance against cationic antimicrobial peptides (e.g., protamine) in the presence of divalent cations (Ca 2þ , Mg 2þ ). Therefore, for the development of peptide-based antibiotics (4), it is important to understand the molecular mode of action of antimicrobial peptides.A number of theoretical models for the interactions of LPS molecules with divalent cations (5-7), suggested that the ions would bind to the charged 2-keto-3-deoxyoctonoic acid (KDO) groups (the "inner core") thereby stabilizing the membrane. Recently, we measured grazing-incidence X-ray scattering from a monolayer of rough mutant LPS from Salmonella enterica sv. Minnesota at an air/water interface and demonstrated the Ca 2þ -induced increase in the electron density near the inner core (8). These observations were supported by the results of Monte Carlo (MC) simulations of a coarse-grained model (8). A further challenge would be to extend such a strategy to wild-type LPSs that possess polydisperse, specific O-polysaccharide chains (O-side chains). Pink et al. (9) carried out MC simulations of a minimal model of the more complex, wild-type LPSs from Pseudomonas aeruginosa (PAO1) and concluded that divalent cations would induce a collapse of the negatively charged O-sidechains toward the membrane surface. Because it is difficult in practice to deposit LPSs wit...