The interactions of two highly positively charged short peptide sequences with negatively charged 
lipid bilayers were explored by fluorescence binding assays and all-atom molecular dynamics 
simulations. The bilayers consisted of mixtures of phosphatidylglycerol (PG), phosphatidylcholine 
(PC) lipids as well as a fluorescence probe that was sensitive to the interfacial potential. The 
first peptide contained nine arginine repeats (Arg9) and the second one had nine lysine repeats 
(Lys9). The experimentally determined apparent dissociation constants and Hill cooperativity 
coefficients demonstrated that the Arg9 peptides exhibited weakly anti-cooperative binding 
behavior at the bilayer interface at lower PG concentrations, but this anti-cooperative effect 
vanished once the bilayers contained at least 20 mol% PG. By contrast, Lys9 peptides showed 
strongly anti-cooperative binding behavior at all PG concentrations and the dissociation constants 
with Lys9 were approximately two orders of magnitude higher than with Arg9. Moreover, only 
arginine rich peptides could bind to the phospholipid bilayers containing just phosphatidylcholine 
lipids. These results along with the corresponding molecular dynamics simulations suggested two 
important distinctions between the behavior of Arg9 and Lys9 that led to these striking differences 
in binding and cooperativity. First, the interactions of the guanidinium moieties on the Arg side 
chains with the phospholipid head groups were stronger than for the amino group. This helped 
facilitate stronger Arg9 binding at all PG concentrations that were tested. However, at PG 
concentrations of 20 mol% or greater, the Arg9 peptides came into sufficiently close proximity 
with each other that favorable like charge pairing between the guanidinium moieties could just 
offset the long range electrostatic repulsions. This led to Arg9 aggregation at the bilayer 
surface.  By contrast, Lys9 molecules experienced electrostatic repulsion from each other at 
all concentrations. These insights may help explain the propensity for cell penetrating peptides 
containing arginine to more effectively cross cell membranes in comparison with lysine rich peptides.