SPIN TRAPPING THE OXIDIZED PRODUCTS OF PUFA IN MODEL MEMBRANES: THE PROTECTION CONFERRED BY VITAMIN E

poster abstractElectron paramagnetic resonance (EPR) spectroscopy is recognized as the most sensitive and noninvasive means to quantify free radicals of biological relevance such as reactive oxygen species (ROS). In spin trapping a molecule (the spin trap) reacts with the free radical producing a spin adduct that is sufficiently stable to be detected by EPR. Here we apply a novel spin trapping technique to investigate the protection that α-tocopherol (vitamin E), the major lipid soluble antioxidant in membranes, confers on polyunsaturated lipids in model membranes. Polyunsaturated fatty acids (PUFA) readily oxidize because they have a cis,cis-1,4-pentadiene motif that renders the central methylene group vulnerable to attack by ROS. Our method quantifies the oxidized products of PUFA in lipid vesicles that have been exposed to a peroxyl radical generator 2,2'-azobis-(amidinopropane) dihydrochloride (AAPH) that initiates the free radical chain reaction. By measuring the reduction in lipid peroxidation due to the presence of αtocopherol, we test the hypothesis that the vitamin co-localizes with polyunsaturated lipids in membrane domains to ensure close proximity to the most vulnerable lipid species

AFFINITY OF CHOLESTEROL FOR POLYUNSATURATED FATTY ACID-CONTAINING PHOSPHOLIPIDS

poster abstractA wide range of health benefits is associated with the consumption of omega-3 polyunsaturated fatty acids (PUFAs). One possible mechanism is that through our diet, they are incorporated into the phospholipids of the plasma membrane and disrupt the molecular organization of membrane do-mains due to the high disorder of PUFA. Our focus is the interaction of PUFA with cholesterol, a major component in plasma membranes. The objective here is to measure the affinity of cholesterol for PUFA-containing phospholip-ids by observing how cholesterol partitions between large unilamellar vesi-cles (LUVs) and Cyclodextrin (CD). Crucial to this determination, we need to be able to determine the concentration of cholesterol in LUVs and CD using an enzymatic colorimetric assay to create a standard curve of light absorb-ance (at 570nm wavelength) as a function of cholesterol concentration. The assay and its application to measuring binding coefficients for cholesterol will be described

Cholesterol\u27s location in lipid bilayers

It is well known that cholesterol modifies the physical properties of lipid bilayers. For example, the much studied liquid-ordered Lo phase contains rapidly diffusing lipids with their acyl chains in the all trans configuration, similar to gel phase bilayers. Moreover, the Lo phase is commonly associated with cholesterol-enriched lipid rafts, which are thought to serve as platforms for signaling proteins in the plasma membrane. Cholesterol\u27s location in lipid bilayers has been studied extensively, and it has been shown – at least in some bilayers – to align differently from its canonical upright orientation, where its hydroxyl group is in the vicinity of the lipid–water interface. In this article we review recent works describing cholesterol\u27s location in different model membrane systems with emphasis on results obtained from scattering, spectroscopic and molecular dynamics studies

SOLUTION STRUCTURE OF THE TOXIC E. COLI PEPTIDE, TISB

poster abstractAntibiotics act by interfering in bacterial metabolism. Thus, antibiotics are only effective against metabolically active bacteria while dormant cells are highly tolerant to antibiotics. Such persistent bacterial cells may be the main culprits in chronic infectious diseases resistance to antimicrobial thera-py. In Escherichia coli, expression of a toxic peptide, TisB, sends cells into dormancy by decreasing the proton motive force thus decreasing ATP levels. TisB is a 29 amino acid residue peptide with 70% hydrophobic residues. It has a predicted alpha helical transmembrane domain spanning residues 6 - 28. In membrane channel studies, ion transport is observed with TisB and with some TisB mutants. As a preliminary to combining multi-dimensional NMR spectroscopy with circular dichroism to determine the structure of the TisB membrane ion transport complex in lipid micelles, NMR spectroscopy is used to determine the structure of TisB in ethanol

Cholesterol shows preference for the interior of polyunsaturated lipid

Recent neutron scattering experiments showed a striking manifestation of the aversion between polyunsaturated fatty acid (PUFA)-containing lipids and cholesterol. Selectively deuterated cholesterol/1,2-diarachidonylphosphabdylcholine (DAPC) samples revealed that the hydroxyl of the sterol resides at the center of the bilayer. Here we use a recently parametrized coarse grain simulation model to shed light on these puzzling experimental observations. Using a simulation setup in close correspondence to the experimental conditions, we reproduce the experimental neutron scattering profiles to a large extent. The simulations allow us to analyze the behavior of cholesterol in detail; we show that the interaction of cholesterol with the PUFA chains of DAPC leads to a fast flip-flop rate for the sterol and an increased preference of the sterol for the unusual location embedded between the monolayer leaflets

Membrane Disordering by Eicosapentaenoic Acid in B Lymphomas Is Reduced by Elongation to Docosapentaenoic Acid as Revealed with Solid-State Nuclear Magnetic Resonance Spectroscopy of Model Membranes

BACKGROUND: Plasma membrane organization is a mechanistic target of n-3 (ω-3) polyunsaturated fatty acids. Previous studies show that eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) differentially disrupt plasma membrane molecular order to enhance the frequency and function of B lymphocytes. However, it is not known whether EPA and DHA affect the plasma membrane organization of B lymphomas differently to influence their function. OBJECTIVE: We tested whether EPA and DHA had different effects on membrane order in B lymphomas and liposomes and studied their effects on B-lymphoma growth. METHODS: B lymphomas were treated with 25 μmol EPA, DHA, or serum albumin control/L for 24 h. Membrane order was measured with fluorescence polarization, and cellular fatty acids (FAs) were analyzed with GC. Growth was quantified with a viability assay. (2)H nuclear magnetic resonance (NMR) studies were conducted on deuterated phospholipid bilayers. RESULTS: Treating Raji, Ramos, and RPMI lymphomas for 24 h with 25 μmol EPA or DHA/L lowered plasma membrane order by 10-40% relative to the control. There were no differences between EPA and DHA on membrane order for the 3 cell lines. FA analyses revealed complex changes in response to EPA or DHA treatment and a large fraction of EPA was converted to docosapentaenoic acid (DPA; 22:5n-3). NMR studies, which were used to understand why EPA and DHA had similiar membrane effects, showed that phospholipids containing DPA, similar to DHA, were more ordered than those containing EPA. Finally, treating B lymphomas with 25 μmol EPA or DHA/L did not increase the frequency of B lymphomas compared with controls. CONCLUSIONS: The results establish that 25 μmol EPA and DHA/L equally disrupt membrane order and do not promote B lymphoma growth. The data open a new area of investigation, which is how EPA's conversion to DPA substantially moderates its influence on membrane properties

DHA Alters Raft-like Membrane Domains as Revealed by Solid State 2H NMR Spectroscopy

poster abstractDietary omega-3 polyunsaturated fatty acids (n-3 PUFAs), such as docosahexaenoic acid (DHA, 22:6), are correlated with the prevention of neurological and autoimmune disorders in humans. These fatty acids must be obtained from the diet, such as oil fish or fish oil supplements, as they cannot be generated within the human body. The origin of the health benefits at the molecular level is still under question. A membrane-mediated mechanism in which n-3 PUFAs are incorporated into phospholipids and modulate molecular organization is one possibility. Cellular membranes are inhomogeneous where structurally diverse lipids can exist in separate domains. Regions rich in sphingomyelin (SM) and cholesterol, commonly called lipid rafts, contain important signaling proteins. In a recent solid-state 2H nuclear magnetic resonance (2H NMR) study of a model membrane composed of 1-[2H31] palmitoyl-2-docosahexaenoyl-phosphatidylcholine (PDPC-d31), a deuterated analog of a DHA-containing phospholipid, in mixtures with SM and cholesterol, we discovered that DHA could significantly enter raft-like domains. How DHA affects the molecular organization within the raft-like domains is addressed here by observing PSM-d31, an analog of SM with a perdeuterated N-palmitoyl chain. The 2H NMR spectra for PSM-d31, in mixtures with PDPC and cholesterol, exhibit two spectral components, a larger more ordered component that we attribute to raft-like domains and a smaller less ordered component that we attribute to non-raft-like domains. On average, the order of PSM-d31 is reduced and, thus, disordering of PSM-d31 by PDPC is indicated. Our observations confirm that DHA can infiltrate rafts and affect molecular organization, which has implications for the signaling of raft and non-raft proteins. Furthermore, these results are consistent with in vivo studies showing that DHA infiltrates rafts

Raft Busters: A Molecular Role for DHA in Biological Membranes?

poster abstractDietary consumption of fish oils rich in omega-3 polyunsaturated fatty acids (n-3 PUFAs), such as docosahexaenoic acid (DHA, 22:6), has a wide variety of health benefits. However, a complete molecular mechanism is yet to be elucidated. One model that has emerged from biochemical and imaging studies of cells postulates that n-3 PUFAs are taken up into phospholipids in the plasma membrane of cells and, due to their high disorder and aversion for cholesterol, reorganize lipid rafts. Lipid rafts are ordered domains within biological membranes which contain high amounts of sphingomyelin (SM) and cholesterol. To investigate this model, we studied lipid bilayers composed of SM, PDPC (a DHA-containing phospholipid), and cholesterol (1:1:1 mol). The molecular organization of each lipid was investigated with solid-state 2H NMR using deuterated analogs of the lipids. Spectral components assigned to ordered raft-like domains and disordered non-raft domains were resolved, from which the composition of the domains and the order within them could be determined. Most of the SM (84%) and cholesterol (88%) was found in the raft-like domain, together with a substantial amount of PDPC (70%). Despite the infiltration of PDPC there appears to be minimal effect on the order of SM or cholesterol. We speculate that PDPC molecules sequester into small groups minimizing the contact of DHA chains with cholesterol, thereby interrupting the continuity of the raft-like environment

DHA and EPA Interaction with Raft Domains Observed With Solid-State 2H NMR Spectroscopy

poster abstractResearch continues to examine the health benefits of omega-3 polyunsaturated fatty acids (n-3 PUFA) found in fish oils. The major bioactive components are eicosapentaenoic acid (EPA, 20:5), with 20 carbons and 5 double bonds, and docosahexaenoic acid (DHA, 22:6), with 22 carbons and 6 double bonds. However, their molecular modes of action remain unclear. A suggested hypothesis is that these fatty acids are incorporated into membrane phospholipids and modify the structure and organization of lipid rafts, thus affecting cell signaling. We used solid-state 2H NMR spectroscopy to compare molecular organization in mixtures of 1-palmitoyl-2-eicosapentaenoylphosphatidylcholine (PEPC) and 1-palmitoyl-2-docosahexaenoylphosphatidylcholine (PDPC) with the raft-stabilizing molecules sphingomyelin (SM) and cholesterol. Our spectra for PEPC-d31 and PDPC-d31, analogs of PEPC and PDPC with a perdeuterated palmitoyl sn-1 chain, showed that DHA has a greater tendency than EPA to incorporate into raft-like domains enriched in SM and cholesterol. By using PSM-d31, an analog of SM with a perdeuterated N-palmitoyl chain, we now directly observe one of the raft-forming molecules and analyze the molecular order within the raft. These results will add to the growing information on how EPA and DHA differentially modify lipid domain organization in bilayers

Vitamin E - phosphatidylethanolamine interactions in mixed membranes with sphingomyelin: Studies by 2H NMR

Among the structurally diverse collection of lipids that comprise the membrane lipidome, polyunsaturated phospholipids are particularly vulnerable to oxidation. The role of α-tocopherol (vitamin E) is to protect this influential class of membrane phospholipid from oxidative damage. Whether lipid-lipid interactions play a role in supporting this function is an unanswered question. Here, we compare the molecular organization of polyunsaturated 1-[2H31]palmitoyl-2-docosahexaenoylphosphatidylethanolamine (PDPE-d31) and, as a control, monounsaturated 1-[2H31]palmitoyl-2-oleoylphosphatidylethanolamine (POPE-d31) mixed with sphingomyelin (SM) and α-tocopherol (α-toc) (2:2:1 mol) by solid-state 2H NMR spectroscopy. In both cases the effect of α-toc appears similar. Spectral moments reveal that the main chain melting transition of POPE-d31 and PDPE-d31 is broadened beyond detection. A spectral component attributed to the formation of inverted hexagonal HII phase in coexistence with lamellar Lα phase by POPE-d31 (20 %) and PDPE-d31 (18 %) is resolved following the addition of α-toc. Order parameters in the remaining Lα phase are increased slightly more for POPE-d31 (7%) than PDPE-d31 (4%). Preferential interaction with polyunsaturated phospholipid is not apparent in these results. The propensity for α-toc to form phase structure with negative curvature that is more tightly packed at the membrane surface, nevertheless, may restrict the contact of free radicals with lipid chains on phosphatidylethanolamine molecules that accumulate polyunsaturated fatty acids