Abstract
We recently described a novel antimicrobial peptide, RTA3, derived from the commensal organism Streptococcus mitis, with strong anti-Gram-negative activity, low salt sensitivity, and minimal mammalian cell toxicity in vitro and in vivo. This peptide conforms to the positively charged, amphipathic helical peptide motif, but has a positively charged amino acid (Arg-5) on the nonpolar face of the helical structure that is induced upon membrane binding. We surmised that disruption of the hydrophobic face with a positively charged residue plays a role in minimizing eukaryotic cell toxicity, and we tested this using a mutant with an R5L substitution. The greatly enhanced toxicity in the mutant peptide correlated with its ability to bind and adopt helical conformations upon interacting with neutral membranes; the wild type peptide RTA3 did not bind to neutral membranes (binding constant reduced by at least 1000-fold). Spectroscopic analysis indicates that disruption of the hydrophobic face of the parent peptide is accommodated in negatively charged membranes without partial peptide unfolding. These observations apply generally to amphipathic helical peptides of this class as we obtained similar results with a peptide and mutant pair (Chen, Y., Mant, C. T., Farmer, S. W., Hancock, R. E., Vasil, M. L., and Hodges, R. S. (2005) J. Biol. Chem. 280, 12316-12329) having similar structural properties. In contrast to previous interpretations, we demonstrate that these peptides simply do not bind well to membranes (like those of eukaryotes) with exclusively neutral lipids in their external bilayer leaflet. We highlight a significant role for tryptophan in promoting binding of amphipathic helical peptides to neutral bilayers, augmenting the arsenal of strategies to reduce mammalian toxicity in antimicrobial peptides.
Highlights
For the strongly partitioning peptides, Kx is in the range of 2.4 ϫ 106 to 1.5 ϫ 107, whereas for the weakly binding systems these are 2 orders of magnitude or more smaller
The binding and membrane-disrupting effects of V681 and RTA3 peptides on PC and PC:PC (50:50) membrane vesicles broadly correlate with their effects on mammalian and bacterial (P. aeruginosa) cells, respectively. To determine whether these observations apply to other lipid compositions, we measured RTA3-induced membrane binding and trapped dye release using 100 nm SUV having lipid compositions more similar to bacterial and mammalian membranes
This study illustrates that vesicle bilayer membranes composed either of neutral lipids or mixed PG:PC membranes can be surprisingly good analogs for eukaryotic and bacterial cell membranes, respectively, consistent with a body of work that highlights membrane surface charge as a dominant feature in the selectivity of positively charged amphipathic peptides for bacterial membranes (5, 30 –33)
Summary
Peptide Synthesis, Purification, and Characterization—The peptides listed in Table 1 were synthesized by Dr G. The fluorescence emission intensity was measured 3 min after mixing CF-loaded vesicles with peptide. Peptide helix content was calculated from the ellipemission spectra of tryptophan in Trp-containing peptides, a 2 ticity at 222 nm (222) (23) using parameters determined by Luo M peptide solution was incubated in buffer A, and aliquots of and Baldwin (24). Peptide binding to FPE-labeled vesicles was measured by adding aliquots of peptide to a suspension of vesicles at 65 M total lipid concentration in buffer A. The experiments were made by adding successive aliquots of peptide to a single vesicle sample. Control experiments showed that the same FPE fluorescence enhancement was obtained by adding a single large aliquot of peptide or the same amount of peptide in successive
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