The effects of gramicidin on the structure of phospholipid assemblies /

Gramicidin is an antibiotic peptide that can be incorporated into the monolayers of cell membranes. Dimerization through hydrogen bonding between gramicidin monomers in opposing leaflets of the membrane results in the formation of an iontophoretic channel. Surrounding phospholipids, with various associated mechanical properties, have been shown to influence the gating properties of this channel. Conversely, gramicidin incorporation has been shown to affect the structure of spontaneously formed lipid assemblies. Using small-angle x-ray diffraction and model systems composed of phospholipids and gramicidin, the physical effects incurred by gramicidin incorporation were measured. The reverse hexagonal (H^) phase composed of dioleoylphosphatidylethanolamine (DOPE) monolayers decreased in lattice dimension with increasing incorporation of gramicidin. This indicated that gramicidin was adding negative curvature to the monolayers. In this system, gramicidin was measured to have an apparent intrinsic radius of curvature (Rop*™") of -7. 1 A. The addition of up to 4 mol% gramicidin in mixtures with DOPE did not result in the monolayers becoming stiffer, as indicated by unaltered bending moduli for each composition. Dioleoylphosphatidylcholine (DOPC) alone forms the lamellar (LJ phase when hydrated, but undergoes a transition into the H^ phase when mixed with gramicidin. The lattice repeat dimension decreases systematically with increased gramicidin content. Again, this indicated that gramicidin was adding negative curvature to the monolayers. At 12 mol% gramicidin in mixtures with DOPC, the apparent radius of intrinsic curvature of gramicidin (Rop*"^) was measured to be -7.4 A. This mixture formed monolayers that were very resistant to bending under osmotic pressure, with a measured bending modulus of 1 15 kT. The measurements made in this study demonstrate that peptides are able to modulate the spontaneous curvature and other mechanical properties of phospholipid assemblies.

Elucidation of a mechanism of cell lysis by chorhexidine : a biophysical approach

Chlorhexidine is an effective antiseptic used widely in disinfecting products (hand soap), oral products (mouthwash), and is known to have potential applications in the textile industry. Chlorhexidine has been studied extensively through a biological and biochemical lens, showing evidence that it attacks the semipermeable membrane in bacterial cells. Although extremely lethal to bacterial cells, the present understanding of the exact mode of action of chlorhexidine is incomplete. A biophysical approach has been taken to investigate the potential location of chlorhexidine in the lipid bilayer. Deuterium nuclear magnetic resonance was used to characterize the molecular arrangement of mixed phospholipid/drug formulations. Powder spectra were analyzed using the de-Pake-ing technique, a method capable of extracting both the orientation distribution and the anisotropy distribution functions simultaneously. The results from samples of protonated phospholipids mixed with deuterium-labelled chlorhexidine are compared to those from samples of deuterated phospholipids and protonated chlorhexidine to determine its location in the lipid bilayer. A series of neutron scattering experiments were also conducted to study the biophysical interaction of chlorhexidine with a model phospholipid membrane of DMPC, a common saturated lipid found in bacterial cell membranes. The results found the hexamethylene linker to be located at the depth of the glycerol/phosphate region of the lipid bilayer. As drug concentration was increased in samples, a dramatic decrease in bilayer thickness was observed. Differential scanning calorimetry experiments have revealed a depression of the DMPC bilayer gel-to-lamellar phase transition temperature with an increasing drug concentration. The enthalpy of the transition remained the same for all drug concentrations, indicating a strictly drug/headgroup interaction, thus supporting the proposed location of chlorhexidine. In combination, these results lead to the hypothesis that the drug is folded approximately in half on its hexamethylene linker, with the hydrophobic linker at the depth of the glycerol/phosphate region of the lipid bilayer and the hydrophilic chlorophenyl groups located at the lipid headgroup. This arrangement seems to suggest that the drug molecule acts as a wedge to disrupt the bilayer. In vivo, this should make the cell membrane leaky, which is in agreement with a wide range of bacteriological observations.