Lipopolysaccharides (LPSs) of different complexities are found on the external bilayer of Gram-negative bacteria. These are postulated to be the target of cationic antibacterial peptides (CAPs). Microbiologists found in in vivo experiments that Ca++ protects bacteria against cationic antibacterial peptides like protamine, which was supported qualitatively by recent theoretical simulations. To fill the gap between the in vivo observation and the theoretical simulations, we search for the structural basis of the function of LPSs on a molecular level. In this study, we focus on the structural characterization of defined mutant LPS?s by a combination of grazing incidence X-ray diffraction (GID) and grazing incidence small-angle X-ray scattering (GISAXS). GID is a powerful technique to resolve in-plane structures of molecular films at a high spatial resolution, such as lattice parameters, and molecular tilt, while GISAXS yields (quasi-) specular reflectivity and thus out-of-plane electron density profiles. We found that the addition of protamine in the absence of calcium induces an increase in surface pressure and a change in both the electron density profile and the in-plane chain ordering. On the other hand, we proove that the presence of Ca++ effectively avoids the protamine insertion. The obtained results offer a direct structural evidence to account for the survival of bacteria with an aid of the carbohydrate-stealth.
