1-palmitoyl-2-oleoylphosphatidylethanolamine and 1-2-dioleoyl-sn-glycero-3-phosphoglycerol

We addressed the onset of synergistic activity of the two well-studied antimicrobial peptides magainin 2 (MG2a) and PGLa using lipid-only mimics of Gram-negative cytoplasmic membranes. Specifically, we coupled a joint analysis of small-angle x-ray and neutron scattering experiments on fully hydrated lipid vesicles in the presence of MG2a and L18W-PGLa to all-atom and coarse-grained molecular dynamics simulations. In agreement with previous studies, both peptides, as well as their equimolar mixture, were found to remain upon adsorption in a surface-aligned topology and to induce significant membrane perturbation, as evidenced by membrane thinning and hydrocarbon order parameter changes in the vicinity of the inserted peptide. These effects were particularly pronounced for the so-called synergistic mixture of 1:1 (mol/mol) L18W-PGLa/MG2a and cannot be accounted for by a linear combination of the membrane perturbations of two peptides individually. Our data are consistent with the formation of parallel heterodimers at concentrations below a synergistic increase of dye leakage from vesicles. Our simulations further show that the heterodimers interact via salt bridges and hydrophobic forces, which apparently makes them more stable than putatively formed antiparallel L18W-PGLa and MG2a homodimers. Moreover, dimerization of L18W-PGLa and MG2a leads to a relocation of the peptides within the lipid headgroup region as compared to the individual peptides. The early onset of dimerization of L18W-PGLa and MG2a at low peptide concentrations consequently appears to be key to their synergistic dye-releasing activity from lipid vesicles at high concentrations.

Topics: Antimicrobial Cationic Peptides; Cell Membrane; Dimerization; Lipid Bilayers; Lipids; Magainins; Molecular Dynamics Simulation; Phosphatidylethanolamines; Phosphatidylglycerols; Temperature

Single-amino acid mutations in the human α-synuclein (αS) protein are related to early onset Parkinson's disease (PD). In addition to the well-known A30P, A53T, and E46K mutants, recently a number of new familial disease-related αS mutations have been discovered. How these mutations affect the putative physiological function of αS and the disease pathology is still unknown. Here we focus on the H50Q and G51D familial mutants and show that like wild-type αS, H50Q and G51D monomers bind to negatively charged membranes, form soluble partially folded oligomers with an aggregation number of ~30 monomers under specific conditions, and can aggregate into amyloid fibrils. We systematically studied the ability of these isolated oligomers to permeabilize membranes composed of anionic phospholipids (DOPG) and membranes mimicking the mitochondrial phospholipid composition (CL:POPE:POPC) using a calcein release assay. Small-angle X-ray scattering studies of isolated oligomers show that oligomers formed from wild-type αS and the A30P, E46K, H50Q, G51D, and A53T disease-related mutants are composed of a similar number of monomers. However, although the binding affinity of the monomeric protein and the aggregation number of the oligomers formed under our specific protocol are comparable for wild-type αS and H50Q and G51D αS, G51D oligomers cannot disrupt negatively charged and physiologically relevant model membranes. Replacement of the membrane-immersed glycine with a negatively charged aspartic acid at position 51 apparently abrogates membrane destabilization, whereas a mutation in the proximal but solvent-exposed part of the membrane-bound α-helix such as that found in the H50Q mutant has little effect on the bilayer disrupting properties of oligomers.

Topics: alpha-Synuclein; Cell Membrane Permeability; Fluoresceins; Humans; Membranes, Artificial; Multiprotein Complexes; Mutation, Missense; Parkinson Disease; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Protein Binding; Scattering, Small Angle; X-Ray Diffraction

We present an overview of the application of dual-focus fluorescence correlation spectroscopy (2f-FCS) for the measurement of diffusion coefficients within free-standing lipid membranes. The first part gives a detailed theoretical analysis of the expected performance of 2f-FCS, in particular about the sensitivity of the method with regard to precise focus position and to aberrations caused by refractive index mismatch or cover slide thickness deviation. After describing the experimental details of the 2f-FCS setup and the preparation of free-standing black lipid membranes (BLMs), we apply the method to study the diffusion of lipids within BLMs as a function of lipid composition and of ion valency and ionic strength of the surrounding buffer.

Topics: Diffusion; Ions; Lipid Bilayers; Lipids; Osmolar Concentration; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Spectrometry, Fluorescence

To investigate the effect of lipid structure upon the membrane topography of hydrophobic helices, the behavior of hydrophobic peptides was studied in model membrane vesicles. To define topography, fluorescence and fluorescence quenching methods were used to determine the location of a Trp at the center of the hydrophobic sequence. For peptides with cationic residues flanking the hydrophobic sequence, the stability of the transmembrane (TM) configuration (relative to a membrane-bound non-TM state) increased as a function of lipid composition on the order: 1:1 (mol:mol) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC):1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine approximately 6:4 POPC:cholesterol

Topics: Amino Acid Sequence; Anions; Cholesterol; Drug Stability; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Membrane Lipids; Membrane Proteins; Membranes, Artificial; Molecular Conformation; Molecular Sequence Data; Peptides; Phosphatidylethanolamines; Phosphatidylglycerols; Phosphatidylserines; Protein Structure, Secondary; Spectrometry, Fluorescence; Static Electricity; Tryptophan