Characterization of the Structure and Membrane Interaction of the Antimicrobial Peptides Aurein 2.2 and 2.3 from Australian Southern Bell Frogs

¹ Dept. Chemistry, University of British Columbia
² Dept. Chemistry, UBC
³ CMDR, University of British Columbia
⁴ Dept. Chem, UBC
⁵ University of British Columbia

^* To whom correspondence should be addressed. E-mail: sstraus{at}chem.ubc.ca.

Submitted on September 12, 2006
Revised on November 2, 2006
Accepted on 4 January 2007

	Abstract

The structure and membrane interaction of the antimicrobial peptide aurein 2.2 (GLFDIVKKVVGALGSL-CONH₂), aurein 2.3 (GLFDIVKKVVGAIGSL-CONH₂), both from Litoria aurea, and a carboxy C-terminal analogue of aurein 2.3 (GLFDIVKKVVGAIGSL-COOH) were studied in order to determine which features of this class of peptides are key to activity. Circular dichroism and solution state NMR data indicate that all three peptides adopt an -helical structure in the presence of trifluoroethanol (TFE) or lipids such as 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and a 1:1 mixture of DMPC and 1,2-dimyristoyl-sn-glycero-3-[Phospho-rac-(1-glycerol)] (DMPG). Oriented circular dichroism was used to determine the orientation of the peptides in lipid bilayers over a range of concentrations (peptide to lipid molar ratios (P/L) = 1:15 to 1:120) in DMPC and 1:1 DMPC/DMPG, in the liquid crystalline state. The results demonstrate that in DMPC all three peptides are surface-adsorbed over a range of low peptide concentrations, but insert into the bilayers at high peptide concentrations. This finding is corroborated by ³¹P solid state NMR data of the three peptides in DMPC, which shows that at high peptide concentrations the peptides perturb the membrane. OCD data of the aurein peptides in 1:1 DMPC/DMPG, on the other hand, shows that the peptides with amidated C-termini readily insert into the membrane bilayers over the concentration range studied (P/L = 1:15 to 1:120), whereas the aurein2.3 peptide with a carboxy C-terminus inserts at a threshold concentration of P/L* between 1:80 and 1:120. Overall, the data presented here suggest that all three peptides studied here interact with PC membranes in a manner which is similar to aurein 1.2 and citropin 1.1, as reported in the literature, with no correlation to the reported activity. On the other hand, both aurein 2.2 and aurein 2.3 behave similarly in PC/PG membranes, while aurein2.3-COOH inserts less readily. As this does not correlate with reported activities, minimal inhibitory concentrations (MIC) of the three peptides against Staph. Aureus (Strain C622, ATCC 25923) and Staph. Epidermidis (Strain C621 - Clinical isolate) were determined. The correlation between structure, membrane interaction, and activity are discussed in light of these results.

Key Words: alpha-helical structure, aurein 2.2 and 2.3, membrane interaction, oriented circular dichroism, solid state NMR, solution state NMR