Solid-State NMR Investigations of Peptide−Lipid Interaction and Orientation of a β-Sheet Antimicrobial Peptide, Protegrin

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Abstract
Protegrin-1 (PG-1) is a broad-spectrum β-sheet antimicrobial peptide found in porcine leukocytes. The mechanism of action and the orientation of PG-1 in lipid bilayers are here investigated using 2H, 31P, 13C, and 15N solid-state NMR spectroscopy. 2H spectra of mechanically aligned and chain-perdeuterated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) bilayers indicate that PG-1 at high concentrations destroys the orientational order of the aligned lamellar bilayer. The conformation of the lipid headgroups in the unoriented region is significantly altered, as seen from the 31P spectra of POPC and the 2H spectra of headgroup-deuterated 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine. These observations indicate that PG-1 disrupts microbial membranes by breaking the extended bilayer into smaller disks, where a significant fraction of lipids is located in the edges of the disks with a distribution of orientations. These edges allow the lipid bilayer to bend back on itself as in toroidal pores. Interestingly, this loss of bilayer orientation occurs only in long-chain lipids such as POPC and not in shorter chain lipids such as 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine (DLPC). To understand the mode of binding of PG-1 to the lipid bilayer, we determined the orientation of PG-1 in DLPC bilayers. The 13CO and 15N chemical shifts of Val-16 labeled PG-1 indicate that the β-strand axis is tilted by 55° ± 5° from the bilayer normal while the normal of the β-sheet plane is 48° ± 5° from the bilayer normal. This orientation favors interaction of the hydrophobic backbone of the peptide with the hydrophobic core of the bilayer and positions the cationic Arg side chains to interact with the anionic phosphate groups. This is the first time that the orientation of a disulfide-stabilized β-sheet membrane peptide has been determined by solid-state NMR.