Peptide:lipid ratio and membrane surface charge determine the mechanism of action of the antimicrobial peptide BP100. Conformational and functional studies

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 a-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 () 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. (C) 2014 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Institut Nacional de Ciencia e Tecnologia de fluidos complexos (INCTFCx)Nude de Apoio Pesquisa de Fluidos Complexos (NAPFCx)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Univ São Paulo, Inst Chem, Dept Biochem, BR-05513970 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, BR-04044020 São Paulo, BrazilUniv Fed Rio de Janeiro, Inst Med Biochem, Nucl Magnet Resonance Natl Ctr, Rio de Janeiro, BrazilEmbrapa Recursos Genet & Biotecnol, BR-70770917 Brasilia, DF, BrazilUniversidade Federal de São Paulo, Dept Biophys, BR-04044020 São Paulo, BrazilFAPESP: 2007/50970-5FAPESP: 2013/08166-5Web of Scienc

The spin label amino acid TOAC and its uses in studies of peptides: chemical, physicochemical, spectroscopic, and conformational aspects

We review work on the paramagnetic amino acid 2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid, TOAC, and its applications in studies of peptides and peptide synthesis. TOAC was the first spin label probe incorporated in peptides by means of a peptide bond. In view of the rigid character of this cyclic molecule and its attachment to the peptide backbone via a peptide bond, TOAC incorporation has been very useful to analyze backbone dynamics and peptide secondary structure. Many of these studies were performed making use of EPR spectroscopy, but other physical techniques, such as X-ray crystallography, CD, fluorescence, NMR, and FT-IR, have been employed. The use of double-labeled synthetic peptides has allowed the investigation of their secondary structure. A large number of studies have focused on the interaction of peptides, both synthetic and biologically active, with membranes. In the latter case, work has been reported on ligands and fragments of GPCR, host defense peptides, phospholamban, and β-amyloid. EPR studies of macroscopically aligned samples have provided information on the orientation of peptides in membranes. More recent studies have focused on peptide–protein and peptide–nucleic acid interactions. Moreover, TOAC has been shown to be a valuable probe for paramagnetic relaxation enhancement NMR studies of the interaction of labeled peptides with proteins. The growth of the number of TOAC-related publications suggests that this unnatural amino acid will find increasing applications in the future

Effect of Counterions on the Shape, Hydration, and Degree of Order at the Interface of Cationic Micelles: the Triflate Case

Specific ion effects in surfactant solutions affect the properties of micelles. Dodecyltrimethylammonium chloride (DTAC), bromide (DTAB), and methanesulfonate (DTAMs) micelles are typically spherical, but some organic anions can induce shape or phase transitions in DTA(+) micelles. Above a defined concentration, sodium triflate (NaTf induces a phase separation in dodecyltrimethylammonium triflate (DTATf micelles, a phenomenon rarely observed in cationic micelles. This unexpected behavior of the DTATf/NaTf system suggests that DTATf aggregates have unusual properties. the structural properties of DTATf micelles were analyzed by time-resolved fluorescence quenching, small-angle X-ray scattering, nuclear magnetic resonance, and electron paramagnetic resonance and compared with those of DTAC, DTAB, and DTAMs micelles. Compared to the other micelle types, the DTATf micelles had a higher average number of monomers per aggregate, an uncommon disk-like shape, smaller interfacial hydration, and restricted monomer chain mobility. Molecular dynamic simulations supported these observations. Even small water-soluble salts can profoundly affect micellar properties; our data demonstrate that the -CF3 group in Tf- was directly responsible for the observed shape changes by decreasing interfacial hydration and increasing the degree of order of the surfactant chains in the DTATf micelles.GENFIT programFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Instituto Nacional de Ciencia e Tecnologia de Fluidos Complexos (INCT-FCx)Nucleo de Apoio a Pesquisa de Fluidos Complexos (NAP-FCx)Univ São Paulo, Inst Quim, São Paulo, BrazilUniv São Paulo, Inst Fis, BR-01498 São Paulo, BrazilUniv Regensburg, Inst Phys & Theoret Chem, D-93053 Regensburg, GermanyUniversidade Federal de São Paulo, Dept Biofis, São Paulo, BrazilUniv Fed ABC, Ctr Ciencias Nat & Humanas, Santo Andre, BrazilUniv São Paulo, FFCLRP, Dept Quim, BR-14049 Ribeirao Preto, BrazilUniversidade Federal de São Paulo, Dept Biofis, São Paulo, BrazilFAPESP: 2007/50970-5FAPESP: 2008/50041-7Web of Scienc