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Protamine is a cationic antimicrobial peptide, which inhibits or kills a number of Gram-negative bacteria, including Pseudomonas aeruginosa and Escherichia coli. Electrostatic interactions between the outer leaflet of the membrane and protamine are thought to be important for the antimicrobial effect. We hypothesized that divalent ions would compete with protamine for binding to the charged O-sidechain of the liposaccharide and expel protamine from the O-sidechains. Experimentally it was shown that increasing concentrations of divalent cations (Ca2+ and Mg2+) reduced the antimicrobial effect of protamine on P. aeruginosa PA01 and E. coli. We also modeled the electrostatic interactions between five protamine Y1 molecules from Atlantic herring and the surface of a Gram-negative bacterium possessing charged O-sidechains of the B-band lipopolysaccharides of P. aeruginosa PA01 in the presence/absence of calcium ions in an aqueous solution described by linearized Poisson−Boltzmann theory with Debye screening lengths κ-1 of 1.0 nm (∼100 mM) and 3.33 nm (∼10 mM). Our conclusions are as follows. [1] A high concentration of calcium ions brought about a slight polysaccharide chain collapse. The calcium ions formed dynamic bridges between the negatively charged O-sidechains on time scales comparable to that of polymer motion. [2] Without the presence of added calcium, all five protamine molecules were trapped in the charged polysaccharide O-sidechain. The probability of finding segments of protamine molecules closer than ∼0.5 nm to the membrane plane (the x−y plane at z = 0) was effectively zero. [3] Both the calcium distribution and the protamine distribution, when present separately, were essentially independent of monovalent ionic concentration for both values of κ. [4] When calcium and protamine were present simultaneously, the effects depended strongly upon the monovalent ion concentration. Added calcium effectively prevented protamine from entering the O-sidechain brush. Conclusions 3 and 4 show that the minimum inhibitory concentration should depend on monovalent ion concentration since complex growth media contain multivalent ions. [5] Our experiments confirmed our theoretical prediction that the addition of Ca2+ significantly reduced the inhibitory effect of protamine. This also confirmed the importance of electrostatic interactions during the first step in protamine's antibacterial mode of action.