title: Peptide:lipid ratio and membrane surface charge determine the mechanism of action of the antimicrobial peptide BP100. Conformational and functional studies.
abstract: The cecropin-melittin hybrid antimicrobial peptide BP100 (H-KKLFKKILKYL-NH2) is selective for Gram-negative bacteria, negatively charged membranes, and weakly hemolytic. We studied BP100 conformational and functional properties upon interaction with large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs, containing variable proportions of phosphatidylcholine (PC) and negatively charged phosphatidylglycerol (PG). CD and NMR spectra showed that upon binding to PG-containing LUVs BP100 acquires α-helical conformation, the helix spanning residues 3-11. Theoretical analyses indicated that the helix is amphipathic and surface-seeking. CD and dynamic light scattering data evinced peptide and/or vesicle aggregation, modulated by peptide:lipid ratio and PG content. BP100 decreased the absolute value of the zeta potential (ζ) of LUVs with low PG contents; for higher PG, binding was analyzed as an ion-exchange process. At high salt, BP100-induced LUVS leakage requires higher peptide concentration, indicating that both electrostatic and hydrophobic interactions contribute to peptide binding. While a gradual release took place at low peptide:lipid ratios, instantaneous loss occurred at high ratios, suggesting vesicle disruption. Optical microscopy of GUVs confirmed BP100-promoted disruption of negatively charged membranes. The mechanism of action of BP100 is determined by both peptide:lipid ratio and negatively charged lipid content. While gradual release results from membrane perturbation by a small number of peptide molecules giving rise to changes in acyl chain packing, lipid clustering (leading to membrane defects), and/or membrane thinning, membrane disruption results from a sequence of events - large-scale peptide and lipid clustering, giving rise to peptide-lipid patches that eventually would leave the membrane in a carpet-like mechanism.
authors.affiliation: Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, C.P. 26077, 05513-970, Brazil.
authors.forename: Mariana C
authors.initials: MC
authors.lastname: Manzini
authors.affiliation: Department of Biophysics, Federal University of São Paulo, São Paulo, 04044-020, Brazil.
authors.forename: Katia R
authors.initials: KR
authors.lastname: Perez
authors.affiliation: Department of Biophysics, Federal University of São Paulo, São Paulo, 04044-020, Brazil.
authors.forename: Karin A
authors.initials: KA
authors.lastname: Riske
authors.affiliation: Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, C.P. 26077, 05513-970, Brazil.
authors.forename: José C
authors.initials: JC
authors.lastname: Bozelli
authors.affiliation: Nuclear Magnetic Resonance National Center, Institute of Medical Biochemistry, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Talita L
authors.initials: TL
authors.lastname: Santos
authors.affiliation: Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, C.P. 26077, 05513-970, Brazil.
authors.forename: Marcia A
authors.initials: MA
authors.lastname: da Silva
authors.affiliation: Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, C.P. 26077, 05513-970, Brazil.
authors.forename: Greice K V
authors.initials: GK
authors.lastname: Saraiva
authors.affiliation: Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, C.P. 26077, 05513-970, Brazil.
authors.forename: Mario J
authors.initials: MJ
authors.lastname: Politi
authors.affiliation: Nuclear Magnetic Resonance National Center, Institute of Medical Biochemistry, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Ana P
authors.initials: AP
authors.lastname: Valente
authors.affiliation: Nuclear Magnetic Resonance National Center, Institute of Medical Biochemistry, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Fábio C L
authors.initials: FC
authors.lastname: Almeida
authors.affiliation: Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, C.P. 26077, 05513-970, Brazil.
authors.forename: Hernan
authors.initials: H
authors.lastname: Chaimovich
authors.affiliation: Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica, P.O. Box 02372, 70770-917, Brasília, Brazil.
authors.forename: Magali A
authors.initials: MA
authors.lastname: Rodrigues
authors.affiliation: Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica, P.O. Box 02372, 70770-917, Brasília, Brazil.
authors.forename: Marcelo P
authors.initials: MP
authors.lastname: Bemquerer
authors.affiliation: Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, C.P. 26077, 05513-970, Brazil. Electronic address: schreier@iq.usp.br.
authors.forename: Shirley
authors.initials: S
authors.lastname: Schreier
authors.affiliation: Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, C.P. 26077, 05513-970, Brazil. Electronic address: imcuccov@iq.usp.br.
authors.forename: Iolanda M
authors.initials: IM
authors.lastname: Cuccovia
mesh_terms: D023181:Antimicrobial Cationic Peptides / Q000737:chemistry* / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D006090:Gram-Negative Bacteria / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008563:Membrane Lipids / Q000737:chemistry* / Q000378:metabolism*; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D010715:Phosphatidylglycerols / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D055672:Static Electricity
chemical_list: D023181:Antimicrobial Cationic Peptides; D008051:Lipid Bilayers; D008563:Membrane Lipids; D009842:Oligopeptides; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; C540525:lysyl-lysyl-leucyl-phenylalanyl-lysyl-lysyl-isoleucyl-leucyl-lysyl-tyrosyl-leucinamide
keywords: Antimicrobial peptide; BP100; CD; Model membrane leakage; NMR; Zeta potential