Oxidatively stressed mitochondria-mimicking membranes: A molecular insight into their organization during apoptosis.

Mitochondria are crucially involved in the removal of eukaryotic cells by the intrinsic pathway of programmed cell death (apoptosis). The mitochondrion's outer membrane (MOM) is the platform where this pathway takes place. Upon oxidative stress triggering apoptotic action, the MOM undergoes permeabilization and release of cytochrome c, ultimately causing cell death. This membrane perforation is regulated not only by opposing members of the Bcl-2 protein family meeting at the MOM but also actively the membrane itself. Upon oxidative damage, the membrane undergoes severe reorganization causing an increase in cell death-causing apoptotic Bcl-2 proteins. To understand the active role of MOM, we provided a detailed molecular view of its structural and dynamic reorganization upon oxidative stress by solid-state 13C MAS NMR (magic angle spinning nuclear magnetic resonance) accompanied by calorimetric studies. By focusing on MOM-like vesicles doped with oxidized lipid species, direct polarization 13C MAS NMR provided a quantitative overview and identification of all lipid moieties across the membrane. 1H-13C cross polarization and insensitive nuclei enhanced by polarization transfer MAS NMR generated a dynamic - mobile versus restricted - membrane profile. Oxidized phospholipids significantly perturb the structural membrane organization and increase membrane dynamics. These perturbations are not uniformly distributed as the hydrophobic core is reflecting the melting of lipid chains and increase in molecular disorder directly, whereas the interface and headgroup region undergo complex dynamical changes, reflecting increased intra-molecular flexibility of these moieties. These changes are potentially crucial in augmenting pro-apoptotic action of proteins like Bax.

Correlated/non-correlated ion dynamics of charge-neutral ion couples: the origin of ionicity in ionic liquids.

Proton/fluoride spin-lattice (T1) nuclear magnetic relaxation dispersion (NMRD) measurements of 1-butyl-3-methyl-1H-imidazolium hexafluorophosphate, [C4mim][PF6], have been carried out using high field spectrometers and a fast-field-cycling instrument at proton Larmor frequencies ranging from 10 kHz to 40 MHz, at different temperatures. The NMRD profiles are interpreted by means of a simple relaxation model based on the inter- and intra-ionic dipole-dipole relaxation mechanism. Using an atomic molecular-ion dynamic simulation at 323 K the relevant spin dipole-dipole (DD) correlation functions are calculated. The results indicate that the NMRD profiles can be rationalized using intra- and inter-ionic spin DD interactions, however, anions are mainly modulated by ionic reorientation because of temporary correlations with cations, where modulation by translational diffusion plays a minor role. Reorientational dynamics of charge-neutral ion couples (i.e. [C4mim][PF6]) and [C4mim]+ ions are in the nano-second (ns) time range whereas the reorientation of [PF6]- is characterized by a reorientational correlation time in the pico-second (ps) regime. Based on the NMRD profiles we conclude that the main relaxation mechanism for [PF6]- is due to fast internal reorientational motion, a partially averaged F-F intra- and F-H inter-ionic DD coupling as the anion resides in close proximity to its temporary oppositely charged cation partner. The F-T1-NMRD data display a ns dispersion which is interpreted as being due to correlated reorientational modulations resulting from the H-containing charge-neutral ion couple [C4mim][PF6]. The analysis of ionicity is based on the free anion fraction, f, and it increases with temperature with f → 1 at the highest temperatures investigated. The fraction is obtained from the H-F NMRD profiles as correlated-non-correlated dynamics of the ions. The analysis of T1 relaxation rates of C, H, F and P at high fields cannot generally give the fraction of ions but is consistent with the interpretation based on the NMRD profiles with relaxation contributions due to DD-intra and -inter, CSA-intra (and -inter for C), including spin rotation for P. The investigation has led to a description of the mechanics governing ion transport in the title ionic liquid via identification of transient correlated/non-correlated ion dynamics.

Phase diagrams of systems with cationic alpha-helical membrane-spanning model peptides and dioleoylphosphatidylcholine.

Ternary phase diagrams have been constructed of systems with dioleoylphosphatidylcholine (DOPC) and water, and two alpha-helical membrane-spanning model peptides, KKLAKK16[KK(LA)6KK] and KKLAKK20[KK(LA)8KK]. It was found that these peptides induced non-lamellar liquid crystalline phases. The amount of peptide needed for this phase transition depended on the water content and the temperature; and for KKLAKK16, a smaller amount of peptide was needed to induce non-lamellar phases than for KKLAKK20. Both peptides were found to induce an isotropic phase, and KKLAKK16 also induced a reversed hexagonal phase. Both peptides may also reside in a lamellar (L(alpha)) phase. When magic angle spinning (MAS) 31P NMR experiments were performed on samples containing the L(alpha) phase and an isotropic phase, four different isotropic chemical shifts were observed. The isotropic chemical shifts could be assigned to the phases, using spinning sidebands to calculate the chemical shift anisotropy (CSA) corresponding to each isotropic shift. MAS 13C NMR also indicated a difference in the aggregational state of the peptides between the L(alpha) and isotropic phases. The phase diagrams were compared to the phase diagram of a similar model peptide, AWW(LA)5WWA in systems with DOPC and water. It was concluded that the phase behaviour was influenced by both electrostatic interactions between the peptides and the lipid headgroups, and the difference between the hydrophobic length of the peptide and the hydrophobic thickness of the lipid bilayer.