Spin-Label Electron Paramagnetic Resonance Spectroscopy Reveals Effects of Wastewater Filter Membrane Coated with Titanium Dioxide Nanoparticles on Bovine Serum Albumin.

The accumulation of proteins in filter membranes limits the efficiency of filtering technologies for cleaning wastewater. Efforts are ongoing to coat commercial filters with different materials (such as titanium dioxide, TiO2) to reduce the fouling of the membrane. Beyond monitoring the desired effect of the retention of biomolecules, it is necessary to understand what the biophysical changes are in water-soluble proteins caused by their interaction with the new coated filter membranes, an aspect that has received little attention so far. Using spin-label electron paramagnetic resonance (EPR), aided with native fluorescence spectroscopy and dynamic light scattering (DLS), here, we report the changes in the structure and dynamics of bovine serum albumin (BSA) exposed to TiO2 (P25) nanoparticles or passing through commercial polyvinylidene fluoride (PVDF) membranes coated with the same nanoparticles. We have found that the filtering process and prolonged exposure to TiO2 nanoparticles had significant effects on different regions of BSA, and denaturation of the protein was not observed, neither with the TiO2 nanoparticles nor when passing through the TiO2-coated filter membranes.

Ibuprofen in a Lipid Bilayer: Nanoscale Spatial Arrangement.

Ibuprofen is a non-steroidal anti-inflammatory drug (NSAID) with analgesic and antipyretic effects. Understanding the molecular mechanisms of drug interaction with cell membranes is important to improving drug delivery, uptake by cells, possible side effects, etc. Double electron-electron resonance spectroscopy (DEER, also known as PELDOR) provides information on the nanoscale spatial arrangement of spin-labeled molecules. Here, DEER was applied to study (mono-)spin-labeled ibuprofen (ibuprofen-SL) in a bilayer of palmitoyl-oleoyl-sn-glycerophosphocholine (POPC). The results obtained show that the ibuprofen-SL molecules are located within a plane in each bilayer leaflet. At their low molar concentration in the bilayer χ, the found surface concentration of ibuprofen-SL is two times higher than χ, which can be explained by alternative assembling in the two leaflets of the bilayer. When χ > 2 mol%, these assemblies merge. The findings shed new light on the nanoscale spatial arrangement of ibuprofen in biological membranes.

Lipid and protein dynamics of stacked and cation-depletion induced unstacked thylakoid membranes.

Chloroplast thylakoid membranes in plants and green algae form 3D architectures of stacked granal membranes interconnected by unstacked stroma lamellae. They undergo dynamic structural changes as a response to changing light conditions that involve grana unstacking and lateral supramolecular reorganization of the integral membrane protein complexes. We assessed the dynamics of thylakoid membrane components and addressed how they are affected by thylakoid unstacking, which has consequences for protein mobility and the diffusion of small electron carriers. By a combined nuclear and electron paramagnetic-resonance approach the dynamics of thylakoid lipids was assessed in stacked and cation-depletion induced unstacked thylakoids of Chlamydomonas (C.) reinhardtii. We could distinguish between structural, bulk and annular lipids and determine membrane fluidity at two membrane depths: close to the lipid headgroups and in the lipid bilayer center. Thylakoid unstacking significantly increased the dynamics of bulk and annular lipids in both areas and increased the dynamics of protein helices. The unstacking process was associated with membrane reorganization and loss of long-range ordered Photosystem II- Light-Harvesting Complex II (PSII-LHCII) complexes. The fluorescence lifetime characteristics associated with membrane unstacking are similar to those associated with state transitions in intact C. reinhardtii cells. Our findings could be relevant for understanding the structural and functional implications of thylakoid unstacking that is suggested to take place during several light-induced processes, such as state transitions, photoacclimation, photoinhibition and PSII repair.

Ether-linked lipids: Spin-label EPR and spin echoes.

Electron spin echo envelope modulation (ESEEM) and conventional electron paramagnetic resonance (EPR) of site-specifically spin-labelled phospholipids are used to investigate the effect of ether-linked chains on the water-penetration and polarity profiles, as well as the phase behaviour and chain flexibility profiles, of phospholipid membranes. D2O-ESEEM reveals that water exposure of the terminal methyl groups in the interdigitated phase of dihexadecyl phosphatidylcholine (DHPC) is comparable to that of the methylene groups at the polar head-group end of the chains. Similarly, an uniform transmembrane polarity profile is obtained from the dependence of the outer 14N-hyperfine splitting on the spin-label position along the chain in frozen interdigitated DHPC dispersions. Two-component conventional EPR spectra of spin labels at the terminal methyl end of the chain reveal that the intermediate gel phase above the pretransition of DHPC contains components in which the lipid chains are interdigitated. The polarity and chain-flexibility profiles in the fluid Lα-phase of DHPC with ether-linked chains are shifted outwards, towards the polar-apolar interface, as compared with that of dihexadecanoyl phosphatidylcholine (DPPC) with ester-linked chains. Also, the polarity profile of DHPC is shifted upwards, to higher polarities. These differences reflect those in hydrocarbon thickness and area/lipid molecule reported by x-ray diffraction for the Lα-phases of the two lipids.

Comparative study of the interaction of CHAPS and Triton X-100 with the erythrocyte membrane.

The zwitterionic detergent CHAPS, a derivative of the bile salts, is widely used in membrane protein solubilization. It is a "facial" detergent, having a hydrophilic side and a hydrophobic back. The objective of this work is to characterize the interaction of CHAPS with a cell membrane. To this aim, erythrocytes were incubated with a wide range of detergent concentrations in order to determine CHAPS partition behavior, and its effects on membrane lipid order, hemolytic effects, and the solubilization of membrane phospholipids and cholesterol. The results were compared with those obtained with the nonionic detergent Triton X-100. It was found that CHAPS has a low affinity for the erythrocyte membrane (partition coefficient K=0.06mM(-1)), and at sub-hemolytic concentrations it causes little effect on membrane lipid order. CHAPS hemolysis and phospholipid solubilization are closely correlated. On the other side, binding of Triton X-100 disorders the membrane at all levels, and has independent mechanisms for hemolysis and solubilization. Differential behavior was observed in the solubilization of phospholipids and cholesterol. Thus, the detergent resistant membranes (DRM) obtained with the two detergents will have different composition. The behaviors of the two detergents are related to the differences in their molecular structures, suggesting that CHAPS does not penetrate the lipid bilayer but binds in a flat position on the erythrocyte surface, both in intact and cholesterol depleted erythrocytes. A relevant result for Triton X-100 is that hemolysis is not directly correlated with the solubilization of membrane lipids, as it is usually assumed.