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

Abstract

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.

Membrane binding of an acyl-lactoferricin B antimicrobial peptide from solid-state NMR experiments and molecular dynamics simulations

“…The CSAs of POPC (≈ 47 ppm ± 1 ppm) and POPE:POPG (3:1) (≈ 37 ppm ± 1 ppm) are furthermore unchanged in the presence of 1 mol % C6-LfB, indicating little observable perturbation of either the neutral or anionic lipid head groups. These findings support our previous results that acylated and Trp-methylated LfB peptides have little effect on the phosphate head groups of DMPC and DMPC:DMPG (3:1) bilayers even at 4 mol %[12]. Moreover, the MD simulations also show no change.…”

“…The C6-LfB6 peptide was synthesized using solid-phase Fmoc methods on an Applied Biosystems (Foster City, CA) 433A peptide synthesizer using modified FastMoc® chemistry according to methods described in Greathouse et al [12] Peptide purity was assessed by reversed phase HPLC using a 4.6 × 50 mm Zorbax SB-C8 column packed with 3.5 μ l octylsilica (Agilent Technologies, Santa Clara CA), at 1 mL/min using a methanol/water gradient (including 0.1% TFA) from 10% to 60% over 8 min followed by a hold at 60% for 5 min. Chromatograms revealed a single major peak and mass spectrometry confirmed the correct mass (1084); therefore, the peptide was used without further purification.…”

“…Modification of AMPs by N-acylation can be an effective method to increase their interaction with microbial membranes and thereby improve their biological activity[12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22]. We have recently reported that the activity of LfB6 peptides can be enhanced by a combination of N-acylation and Trp-methylation[12].…”

“…We have recently reported that the activity of LfB6 peptides can be enhanced by a combination of N-acylation and Trp-methylation[12]. Here we report on the differential effects of the LfB6 peptide aminoacylated with a 6-carbon chain (C6-LfB6; CH 3 (CH 2 ) 4 CO-RRWQWR-NH 2 ) on model bacterial-like and mammalian-like membranes using all-atom molecular dynamics simulations and solid-state 2 H and 31 P NMR spectroscopy.…”

Cationic Antimicrobial Peptides

“…21A, Tables 3 and 5). Studies with a 1MeW-modified lactoferricin analog revealed enhanced membrane binding and showed 1MeW to be aligned at the membrane interface with an extent of motion similar to that of W [135]. Furthermore, the influence of dipole and quadrupole moments became apparent in intramolecular interactions between the -electrons of the indole ring of W with the positively charged guanidino moiety of R in RW-rich peptides, which were suggested to stabilize the structure of CAPs and enhance membrane binding [136,137].…”