Lipid interactions of acylated tryptophan-methylated lactoferricin peptides by solid-state NMR

Lactoferricin (LfB) is a 25-residue innate immunity peptide released by pepsin from the N-terminal region of bovine lactoferrin. A smaller amidated peptide, LfB6 (RRWQWR-NH₂) retains antimicrobial activity and is thought to constitute the "antimicrobial active-site" (Tomita, Acta Paediatr Jpn. 1994; 36: 585-91). Here we report on N-acylation of 1-Me-Trp⁵-LfB6, Cn-RRWQ[1-Me-W]R-NH2, where Cn is an acyl chain having n = 0, 2, 4, 6 or 12 carbons. Tryptophan 5 (Trp⁵) was methylated to enhance membrane binding and to allow for selective deuteration at that position. Peptide/lipid interactions of Cn-RRWQ[1-Me-W]R-NH₂ (deuterated 1-Me-Trp⁵ underlined), were monitored by solid state ³¹P NMR and 2H NMR. The samples consisted of macroscopically oriented bilayers of mixed neutral (dimyristoylphosphatidylcholine, DMPC) and anionic (dimyristoylphosphatidylglycerol, DMPG) lipids in a 3 : 1 ratio with Cn-RRWQ[1-Me-W]R-NH₂ peptides added at a 1 : 25 peptide to lipid ratio. 2H-NMR spectra reveal that the acylated peptides are well aligned in DMPC:DMPG bilayers. The ²H NMR quadrupolar splittings suggest that the 1-Me-Trp is located in a motionally restricted environment, indicating partial alignment at the membrane interface. ³¹P-NMR spectra reveal that the lipids are predominantly in a bilayer configuration, with little perturbation by the peptides. Methylation alone, in C0-RRWQ[1-Me-W]R-NH₂, resulted in a 3-4 fold increase in antimicrobial activity against E. coli. N-acylation with a C12 fatty acid enhanced activity almost 90 fold.