Membrane Binding, Structure, and Localization of Cecropin-Mellitin Hybrid Peptides: A Site-Directed Spin-Labeling Study

Abstract

The interaction of antimicrobial peptides with membranes is a key factor in determining their biological activity. In this study we have synthesized a series of minimized cecropin-mellitin hybrid peptides each containing a single cysteine residue, modified the cysteine with the sulfhydryl-specific methanethiosulfonate spin-label, and used electron paramagnetic resonance spectroscopy to measure membrane-binding affinities and determine the orientation and localization of peptides bound to membranes that mimic the bacterial cytoplasmic membrane. All of the peptides were unstructured in aqueous solution but underwent a significant conformational change upon membrane binding that diminished the rotational mobility of the attached spin-label. Apparent partition coefficients were similar for five of the six constructs examined, indicating that location of the spin-label had little effect on peptide binding as long as the attachment site was in the relatively hydrophobic C-terminal domain. Depth measurements based on accessibility of the spin-labeled sites to oxygen and nickel ethylenediaminediacetate indicated that at high lipid/peptide ratios these peptides form a single alpha-helix, with the helical axis aligned parallel to the bilayer surface and immersed approximately 5 A below the membrane-aqueous interface. Such a localization would provide exposure of charged/polar residues on the hydrophilic face of the amphipathic helix to the aqueous phase, and allow the nonpolar residues along the opposite face of the helix to remain immersed in the hydrophobic phase of the bilayer. These results are discussed with respect to the mechanism of membrane disruption by antimicrobial peptides.

Interaction of heat shock protein 70 with membranes depends on the lipid environment

“…Similarly, antimicrobial peptides are inserted spontaneously into membranes, forming disruptive pores that :1) were incubated with increasing concentrations of Hsp70 (1-8 μg/ml) for 30 min at 25°C. Individual spectra (386-nm excitation) were acquired, and the maximum peak signal intensity was presented with respect to Hsp70 concentrations appear to gain an α helix conformation upon insertion into the lipid bilayer (Bhargava and Feix 2004). Moreover, penetratin, which is also a random-coil monomer in solution, overcame a conformational change from α-helical to β-like conformations in the presence of lipid membranes, resulting in an aggregation that could be visualized in giant unilamellar vesicles (Lee et al 2010).…”

Anticancer effects of CAMEL peptide

“…It suggests that CAMEL destroys mitochondrial 15 postulated that CAMEL destroys bacterial membrane through a detergent-like 'carpet' mechanism or by channel formation disturbing ion gradient. According to these researchers, in aqueous solutions CAMEL does not adopt any definite structure, whereas upon attaching to the membrane it forms an a-helix that localizes ultimately within the lipid bilayer.…”

Lysine-Enriched Cecropin-Mellitin Antimicrobial Peptides with Enhanced Selectivity

“…In an ␣-helical configuration, CM15 has an almost-ideal amphipathic distribution of amino acid side chains (3,16). Omitting the first two residues (Lys1 Trp2), which are not in a helical configuration in either full-length cecropins (12,19) or a 26-residue cecropin-mellitin hybrid (20), the remaining four lysine residues lie along one surface of the helix, and the opposite face is composed entirely of nonpolar residues (Fig.…”

“…Peptides were synthesized with acetylated N termini and amidated C termini and purified by semipreparative reversephase high-pressure liquid chromatography (RP-HPLC) as described previously (3). Analytical RP-HPLC and mass spectrometry were used to verify purity and composition.…”