Antimicrobial peptides (AMPs) exhibit cell selectivity and activity against microorganisms and are promising candidates as pharmaceutical agents. In mammalian cell membranes cholesterol plays a regulatory function in antibiotic drug resistance and the immune response. Our hypothesis is that differences in the peptide-membrane interactions versus cholesterol affect the bilayer properties giving a possible framework for the selectivity of AMPs for bacterial membranes. Here we employed solid-state 2H NMR to compare the degree of softening or stiffening by AMPs compared to cholesterol in model di-monounsaturated phosphatidylcholine and phosphatidylethanolamine lipids (DOPC versus DOPE). Solid-state 2H NMR quadrupolar-echo experiments and relaxometry experiments (Chem. Phys. Lipids 2016, 1848, 246) investigated changes in structural properties and mechanical properties due to the membrane-peptide interaction. The DOPC and DOPE with LL37 (2-4 mol%) model systems mimicking mammalian and bacterial membranes were studied with POPC-d31 (10 mol%) as a probe lipid. The residual quadrupolar couplings from quadrupolar-echo experiments yielded segmental order parameters (SCD) for the individual acyl segments. Notably LL37 decreased the quadrupolar splittings indicating it requires a specific area per lipid, while cholesterol showed the opposite behavior. The model-free functional dependence of spin-lattice relaxation rates on SCD (square-law plots) indicated greater bending rigidity with cholesterol for both DOPC and DOPE membranes. By contrast, LL37 decreased the bending rigidity of the membranes. Additionally, bilayers comprising POPE/DMPG/cardiolipin (90:5:5), and DMPC:cholesterol (90:10) were modeled with LL37 peptides on the bilayer surface. All-atom molecular dynamics simulation trajectories captured the molecular-level interactions. We observed partial insertion of the LL37 in the POPE system, while the peptides were repelled from the membrane in the DMPC/cholesterol system. These observations reveal the surprising rivalry between cholesterol and AMPs which underlies AMP mechanism and selectivity.

Molecular simulations and NMR spectroscopy reveal antimicrobial peptide membrane interactions

ACS Spring 2022

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