Using infrared spectroscopy of cyanylated cysteine to map the membrane binding structure and orientation of the hybrid antimicrobial peptide CM15

Abstract

The synthetic antimicrobial peptide CM15, a hybrid of N-terminal sequences from cecropin and melittin peptides, has been shown to be extremely potent. Its mechanism of action has been thought to involve pore formation based on prior site-directed spin labeling studies. This study examines four single-site β-thiocyanatoalanine variants of CM15 in which the artificial amino acid side chain acts as a vibrational reporter of its local environment through the frequency and line shape of the unique CN stretching band in the infrared spectrum. Circular dichroism experiments indicate that the placements of the artificial side chain have only small perturbative effects on the membrane-bound secondary structure of the CM15 peptide. All variant peptides were placed in buffer solution, in contact with dodecylphosphatidylcholine micelles, and in contact with vesicles formed from Escherichia coli polar lipid extract. At each site, the CN stretching band reports a different behavior. Time-dependent attenuated total reflectance infrared spectra were also collected for each variant as it was allowed to remodel the E. coli lipid vesicles. The results of these experiments agree with the previously proposed formation of toroidal pores, in which each peptide finds itself in an increasingly homogeneous and curved local environment without apparent peptide-peptide interactions. This work also demonstrates the excellent sensitivity of the SCN stretching vibration to small changes in the peptide-lipid interfacial structure.

Figure 1

Helical wheel diagram for CM15 at the lipid/solvent interface based on previous studies (8, 12), with sites chosen for substitution with cyanylated cysteine in red.

Figure 2

Far-UV circular dichroism for all CM15 peptides in aqueous buffer and in DPC micelle solution. C* locations are marked on each plot.

Figure 3

Infrared CN stretching absorption bands for C*-labeled CM15 variants in aqueous buffer solution, DPC micelles, and BPL solution after 20 minutes of exposure. The non-scaled maximum absorbance of all samples was between 200 and 800 μO.D.

Figure 4

(A)–(D) Infrared CN stretching absorption bands for C*-labeled CM15 variants (with label location marked on each plot) as a function of time in BPL solution. (E) Average frequencies and (F) peak variances for all CN bands as a function of time.

Figure 5

CN stretching band for V14C* in BPL solution after 20 minutes of exposure, fit to two Gaussians with variable widths (w) and frequencies (xc).

Figure 6

Cartoon representation of the positioning of CM15 during pore formation in BPL bilayers, with IR label sites indicated. Graphical distances do not correspond to any physical scale. Light blue=aqueous solution, dark blue=polar head groups, yellow=hydrophobic lipid chains, red cylinder=CM15 helix.