Aggregation and hemi-fusion of anionic vesicles induced by the antimicrobial peptide cryptdin-4.
We show that cryptdin-4 (Crp4), an antimicrobial peptide found in mice, induces the aggregation and hemi-fusion of charged phospholipid vesicles constructed of the anionic lipid POPG and the zwitterionic lipid POPC. Hemi-fusion is confirmed with positive total lipid-mixing assay results, negative inner monolayer lipid-mixing assay results, and negative results from contents-mixing assays. Aggregation, as quantified by absorbance and dynamic light scattering, is self-limiting, creating finite-sized vesicle assemblies. The rate limiting step in the formation process is the mixing of juxtaposed membrane leaflets, which is regulated by bound peptide concentration as well as vesicle radius (with larger vesicles less prone to hemi-fusion). Bound peptide concentration is readily controlled by total peptide concentration and the fraction of anionic lipid in the vesicles. As little as 1% PEGylated lipid significantly reduces aggregate size by providing a steric barrier for membrane apposition. Finally, as stable hemi-fusion is a rare occurrence, we compare properties of Crp4 to those of many peptides known to induce complete fusion and lend insight into conditions necessary for this unusual type of membrane merger.
Cummings
Jason E
JE
Department of Chemical Engineering, Princeton University, Princeton, NJ 08544, USA.
Vanderlick
T Kyle
TK
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives / Q000737:chemistry; D000818:Animals; D008027:Light; D051379:Mice; D010714:Phosphatidylethanolamines / Q000737:chemistry; D010715:Phosphatidylglycerols / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D012235:Rhodamines / Q000737:chemistry; D012542:Scattering, Radiation; D053835:Unilamellar Liposomes / Q000737:chemistry*; D023084:alpha-Defensins / Q000494:pharmacology*
C053685:N-(lissamine rhodamine B sulfonyl)-1,2-dioleoylphosphatidylethanolamine; D010714:Phosphatidylethanolamines; D010715:Phosphatidylglycerols; D012235:Rhodamines; D053835:Unilamellar Liposomes; D023084:alpha-Defensins; C401847:cryptdin 4, mouse; D011092:Polyethylene Glycols; C060037:1-palmitoyl-2-oleoylglycero-3-phosphoglycerol; C077128:N-(7-nitro-2,1,3-benzoxadiazol-4-yl)dioleoylphosphatidylethanolamine; D009327:4-Chloro-7-nitrobenzofurazan