Oxidation of unsaturated lipids in cellular membranes has been shown to cause severe membrane damage and potentially cell death. The presence of oxidized lipid species in the membrane is known to cause changes in membrane properties, such as decreased fluidity. This study uses giant unilamellar vesicles (GUVs) to measure passive transport across membranes containing defined concentrations of oxidized lipid species. GUVs consisting of a saturated phospholipid, an unsaturated phospholipid, and cholesterol were used as model membranes. By replacing defined amounts of the unsaturated lipid with a corresponding oxidized product, the oxidation process could be mimicked, yielding vesicles of varying oxidized lipid concentration. Oxidized lipid concentration was varied from 0 mol% to 18 mol% of the total lipid concentration. Passive transport of PEG12-NBD, an uncharged fluorescent molecule, was measured using a microfluidic trap to capture the GUVs and spinning disk confocal microscopy (SDCM) to track the transport of a fluorescent species in the equatorial plane of each GUV. Membrane permeability was determined by fitting the resulting concentration profiles to a finite difference model of diffusion and permeation around and through the membrane. Experiments showed three permeability regimes. Without oxidation, transport was slow, with a measured permeability on the order of 1.5×10−6 cm/s. At 2.5–10% oxidized species permeation was fast (1.5×10−5 cm/s). Above 12.5% oxidized species, the bilayer was disrupted by the formation of pore defects. As passive transport is an important mechanism for drug delivery, understanding the relationship between oxidation and permeation could provide insight into the pharmaceutical characteristics of tissues with oxidative damage.

Lipid oxidation has been linked to plasma membrane damage leading to cell death. In previous work, we examined the effect of oxidation on bilayer permeability by replacing defined amounts of an unsaturated lipid species with the corresponding phospholipid product that would result from oxidative tail scission of that species. This study adds the cleaved tail fragment, better mimicking the chemical results of oxidation. Permeability of PEG12-NBD, a small, uncharged molecule, was measured for vesicles with oxidation concentration corresponding to between 0 and 18 mol % of total lipid content. Permeability was measured using a microfluidic trap to capture the vesicles and spinning disk confocal microscopy (SDCM) to measure the transport of fluorescent PEG12-NBD at the equatorial plane. The thicknesses of lipid bilayers containing oxidized species were estimated by measuring capacitance of a black lipid membrane while simultaneously measuring bilayer area. We found that relative to chemically modeled oxidized bilayers without tail fragments, bilayers containing cleaved tail groups were less permeable for the same degree of oxidation. Curiously, membrane capacitance measurements indicated that the addition of tail fragments to chemically modeled oxidized bilayers also thinned these bilayers relative to samples with no tail fragments; in other words, the more permeable membranes were thicker. Above 12.5% chemically modeled oxidation, compositions both with and without the cleaved tail groups showed pore formation. This work highlights the complexity of the relationship between chemically modeled lipid bilayer oxidation and cell membrane properties.

phosphate backbone and their impact on the folding free energy, we have formulated a mathematical treatment for computing the volume of the mainchain torsion angle conformation space between every pair of nucleobases along any sequence and the corresponding backbone entropy. We compare the computed conformational entropies against a statistical free energy analysis of structures in the crystallographic database from several thousand backbone conformations between nearest-neighbor nucleobases as well as against computer simulations. Using this calculation we have analyzed the backbone entropy of several ribozymes and riboswitches and found that their entropic strains are highly localized. This suggests the folding and stability of the RNA structures are critically dependent on these local entropic strain domains and leave the rest of the sequence relatively flexible. The total entropic penalty in the backbone for the native fold can be as high as 0.7 cal/K/mol per nucleotide for these medium and large RNAs, producing a contribution to the overall free energy of up to 40 kcal/mol for a 200-nucleotide structure. We also look at the correlation of nucleotide conformations along several sample loop sequences to determine the effect of nucleotides beyond the nearest-neighbor. Oxidation of unsaturated lipids in cellular membranes has been shown to cause severe membrane damage and potentially cell death. Even in low concentrations, oxidized lipid species are known to cause changes in the membrane structure, such as decreased fluidity. Vesicles containing concentrations of oxidized species as low as 20mol% total lipid concentration can display the spontaneous formation of pores. Below this poration limit, the effects of oxidation on membrane permeability have not been quantified. Here, we use giant unilamellar vesicles (GUVs) as a system to measure passive transport across membranes containing defined concentrations of oxidized lipid species. GUVs consisting of a saturated phospholipid, an unsaturated phospholipid, and cholesterol were used as a model membranes. By replacing defined amounts of the unsaturated lipid with a corresponding oxidized product, the oxidation process could be mimicked, yielding vesicles of varying oxidized lipid concentration. Oxidized lipid concentration was varied from 0mol% to 15mol% of the total lipid concentration. We measured passive transport across the membrane using a microfluidic trap to capture the vesicles and spinning disk confocal microscopy to track the transport of a fluorescent species in the equatorial plane of each GUV analyzed. We used fluorescently labeled short-chain poly(ethylene glycol) species of various molecular weights to track the diffusion of a representative small molecule. Membrane permeability was determined by fitting the resulting concentration profiles to a finite element model of diffusion and permeation around and through the membrane. Experiments showed that an increase in oxidized concentration increases membrane permeability. As passive transport is an impo...

in the wheat-germ-extract (WGE) cell-free system, showing that the ring can be formed with the c subunit produced with a WGE cell-free system. Then, solid-state NMR analysis was carried out. Two-dimensional 13 C homonuclear correlation with DARR and 2D Ca(iþ1)-Ca(i) correlation experiments were performed under MAS. The assignment was carried out, using fully 13 C, 15 N labeled TF o c and partially 13 C, 15 N labeled TF o c. The assignment strategy and the obtained structural information on the basis of the assignment and magnetization transfer will be discussed.