Browsing by Author "Williams, Justin A."
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Item AFFINITY OF CHOLESTEROL FOR POLYUNSATURATED FATTY ACID-CONTAINING PHOSPHOLIPIDS(Office of the Vice Chancellor for Research, 2012-04-13) Kagimbi, Maureen W.; Williams, Justin A.; Wassall, Stephen R.A 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.Item Biophysical studies of cholesterol in unsaturated phospholipid model membranes(2013) Williams, Justin A.; Wassall, Stephen R.; Decca, Ricardo; Petrache, Horia; Zhu, Fangqiang; Todd, Brian A.Cellular membranes contain a staggering diversity of lipids. The lipids are heterogeneously distr ibuted to create regions, or domains, whose physical properties differ from the bulk membrane and play an essential role in modulating the function of resident proteins. Many basic questions pertaining to the formation of these lateral assemblies remain. T his research employs model membranes of well - defined composition to focus on the potential role of polyunsaturated fatty acids (PUFAs) and their interaction with cholesterol (chol) in restructuring the membrane environment. Omega - 3 (n - 3) PUFAs are the main bioactive components of fish oil, whose consumption alleviates a variety of health problems by a molecular mechanism that is unclear. We hypothesize that the incorporation of PUFAs into membrane lipids and the effect they have on molecular organization may be, in part, responsible. Chol is a major constituent in the plasma membrane of mammals. It determines the arrangement and collective properties of neighboring lipids, driving the formation of domains via differential affinity for different lipids . T he m olecular organization of 1 -[ 2 H 31 ]palmitoyl -2- eicosapentaenoylphosphatidylcholine (PEPC - d 31 ) and 1 -[ 2 H 31 ]palmitoyl -2- docosahexaenoylphosphatidylcholine (PDPC -d 31 ) in membran es with sphingomyelin (SM) and chol (1:1:1 mol) was compared by solid - state 2 H NMR spectroscopy. Eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids are the two major n - 3 PUFAs found in fish oil, while PEPC -d 31 and PDPC -d 31 are phospholipids containing the respective PUFAs at the sn - 2 position and a perdeuterated palmitic acid a t the sn - 1 position . Analysis of s pectra recorded as a function of temperature indicate s that in both cases, formation of PUFA - rich (less ordered) and SM - rich (more ordered) domains occurred. A surprisingly substantial proportion of PUFA was found to infil trate the more ordered domain. There was almost twice as much DHA (65%) as EPA (30%) . The implication is that n - 3 PUFA s can incorporate into lipid rafts, which are domains enriched in SM and chol in the plasma membrane, and potentially disrupt the activity of signaling proteins that reside therein. DHA, furthermore, may be the more potent component of fish oil. PUFA - chol interactions were also examined through affinity measurements. A novel method utilizing electron paramagnetic resonance (EPR) was develope d, to monitor the partitioning of a spin - labeled analog of chol , 3β - doxyl - 5α - cholestane (chlstn), between large unilamellar vesicles (LUVs) and met hyl - β - cyclodextrin (mβCD). The EPR spectra for chlstn in the two environments are distinguishable due to the substantial differences in tumbling rates , allowing the population distribution ratio to be determined by spectral simulation. Advantages of this approach include speed of implementation and a vo idance of potential artifact s associated with physical separation of LUV and mβCD . Additionally, in a check of the method, t he relative partition coefficients between lipids measured for the spin label analog agree with values obtained for chol by isothermal titration calorimetry (ITC). Results from LUV with different composition confirmed a hierarchy of decreased sterol affinity for phospholipids with increasing acyl chain unsaturation , PDPC possessing half the affinity of the corresponding monounsaturated phospholipid. Taken together, the results of these studies on model membranes demonstrate the potential for PUFA - driven alteration of the architecture of biomembranes, a mechanism through which human health may be impacted.Item DHA Alters Raft-like Membrane Domains as Revealed by Solid State 2H NMR Spectroscopy(Office of the Vice Chancellor for Research, 2015-04-17) Kinnun, Jacob J.; Williams, Justin A.; Stillwell, William; Bittman, Robert; Shaikh, Saame Raza; Wassall, Stephen R.Dietary 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.Item DHA and EPA Interaction with Raft Domains Observed With Solid-State 2H NMR Spectroscopy(Office of the Vice Chancellor for Research, 2013-04-05) Kinnun, Jacob J.; Williams, Justin A.; Stillwell, William H.; Bittman, Robert; Shaikh, Saame Raza; Wassall, Stephen R.Research 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.Item Effects of Lipid Interactions on Model Vesicle Engulfment by Alveolar Macrophages(Elsevier B.V., 2014-02-04) Justice, Matthew J.; Petrusca, Daniela N.; Rogozea, Adriana L.; Williams, Justin A.; Schweitzer, Kelly S.; Petrache, Irina; Wassall, Stephen R.; Petrache, Horia I.; Department of Physics, School of ScienceThe engulfment function of macrophages relies on complex molecular interactions involving both lipids and proteins. In particular, the clearance of apoptotic bodies (efferocytosis) is enabled by externalization on the cell target of phosphatidylserine lipids, which activate receptors on macrophages, suggesting that (local) specific lipid-protein interactions are required at least for the initiation of efferocytosis. However, in addition to apoptotic cells, macrophages can engulf foreign bodies that vary substantially in size from a few nanometers to microns, suggesting that nonspecific interactions over a wide range of length scales could be relevant. Here, we use model lipid membranes (made of phosphatidylcholine, phosphatidylserine, and ceramide) and rat alveolar macrophages to show how lipid bilayer properties probed by small-angle x-ray scattering and solid-state 2H NMR correlate with engulfment rates measured by flow cytometry. We find that engulfment of protein-free model lipid vesicles is promoted by the presence of phosphatidylserine lipids but inhibited by ceramide, in accord with a previous study of apoptotic cells. We conclude that the roles of phosphatidylserine and ceramide in phagocytosis is based, at least in part, on lipid-mediated modification of membrane physical properties, including interactions at large length scales as well as local lipid ordering and possible domain formation.Item Interaction of α-tocopherol with a polyunsaturated lipid studied by MD simulations(Office of the Vice Chancellor for Research, 2013-04-05) Leng, Xiaoling; Williams, Justin A.; Marquardt, Drew; Kučerka, Norbert; Katsaras, John; Atkinson, Jeffrey; Harroun, Thad A.; Feller, Scott E.; Wassall, Stephen R.Polyunsaturated phospholipids are essential components of neural membranes and their effect on membrane architecture is proposed to be the molecular origin of a myriad of health benefits. A downside of polyunsaturated phospholipids is that they are highly susceptible to oxidation due to the presence of multiple double bonds. α-Tocopherol is the most biologically active component in a family of phenolic compounds that comprise vitamin E, which is the major lipid soluble antioxidant in cell membranes. To investigate whether α-tocopherol preferentially interacts with polyunsaturated phospholipids to optimize protection against oxidation, we performed MD simulations on 1-stearoyl-2-docosahexaenoylphosphatiylcholine (SDPC, 18:0-22:6PC) and 1-stearoyl-2-oleoylphosphatidylcholine (SOPC, 18:0-18:1PC) bilayers containing α-tocopherol. SDPC with a docosahexaenoyl sn-2 chain is polyunsaturated, while SOPC with an oleoyl sn-2 chain serves as a monounsaturated control. The simulations were run under constant pressure for 200 ns on a system that comprised 80 phospholipid molecules, 20 α-tocopherol molecules and 2165 water molecules. We discovered significant differences between the two systems. Analysis of the simulations indicates that the α-tocopherol has a strong interaction with the polyunsaturated fatty acid. The flip-flop of α-tocopherol across the bilayer is also much faster in SDPC than in SOPC. Solid state NMR, neutron scattering and complementary experiments are now underway to test the predictions from the MD simulations.Item An Investigation of Whether Vitamin E Preferentially Interacts with Polyunsaturated Lipids(Office of the Vice Chancellor for Research, 2015-04-17) Cavazos, Andres; Kinnun, Jacob J.; Williams, Justin A.; Bank, Morris; Ray, Bruce D.; Harper, Paul E.Vitamin E (α-tocopherol) is a lipid-soluble antioxidant that has the role of protecting phospholipids from oxidation in membranes. A question that remains is how the low concentration of α-tocopherol found in whole cells can protect the relatively large concentration of polyunsaturated phospholipids found in membranes that are particularly vulnerable to oxidative attack. We hypothesize that α-tocopherol colocalizes with polyunsaturated phospholipids to optimize its role as an antioxidant. This project attempts to test this hypothesis by comparing the effect of α-tocopherol on the molecular organization of 1-palmitoyl-2-docosahexaenoyl-sn-glycerophosphatidylethanolamine (16:0-22:6PE, PDPE) and, as a monounsaturated control, 1-palmitoyl-2-oleoyl-sn-glycerophosphatidylethanolamine (16:0-18:1PE, POPE) in mixtures with sphingomyelin (SM). By solid-state 2H NMR spectroscopy, we directly observe order and phase behavior of POPE-d31 and PDPE-d31 (analogs of POPE and PDPE with a perdeuterated sn-1 chain) in the mixed membranes. In complementary X-ray diffraction and differential scanning calorimetry experiments we further probe phase behavior. The spectra observed for POPE-d31 in POPE/SM/α-tocopherol (2:2:1 mol) reveal that a transition from gel to liquid crystalline phase is no longer apparent. At higher temperatures there is a superposition of two spectral components that we ascribe to α-tocopherol promoting a transition from lamellar to inverted hexagonal (HII) phase. Analysis of depaked spectra shows that order is increased by about 8 % and that the amount of HII phase increases with temperature, ranging from 7 (31 °C) to 41 % (65 °C). In mixed membranes where POPE-d31 is replaced by PDPE-d31, we shall investigate whether there is a greater tendency for α-tocopherol to increase order and destabilize bilayer structure for the polyunsaturated phospholipid.Item Lipid bilayer thickness determines cholesterol's location in model membranes(RSC, 2016-12) Marquardt, Drew; Heberle, Frederick A.; Greathouse, Denise V.; Koeppe, Roger E., II.; Standaert, Robert F.; Van Oosten, Brad J.; Harroun, Thad A.; Kinnun, Jacob J.; Williams, Justin A.; Wassall, Stephen R.; Katsaras, John; Department of Physics, School of ScienceCholesterol is an essential biomolecule of animal cell membranes, and an important precursor for the biosynthesis of certain hormones and vitamins. It is also thought to play a key role in cell signaling processes associated with functional plasma membrane microdomains (domains enriched in cholesterol), commonly referred to as rafts. In all of these diverse biological phenomena, the transverse location of cholesterol in the membrane is almost certainly an important structural feature. Using a combination of neutron scattering and solid-state 2H NMR, we have determined the location and orientation of cholesterol in phosphatidylcholine (PC) model membranes having fatty acids of different lengths and degrees of unsaturation. The data establish that cholesterol reorients rapidly about the bilayer normal in all the membranes studied, but is tilted and forced to span the bilayer midplane in the very thin bilayers. The possibility that cholesterol lies flat in the middle of bilayers, including those made from PC lipids containing polyunsaturated fatty acids (PUFAs), is ruled out. These results support the notion that hydrophobic thickness is the primary determinant of cholesterol's location in membranes.Item Raft Busters: A Molecular Role for DHA in Biological Membranes?(Office of the Vice Chancellor for Research, 2016-04-08) Kinnun, Jacob J.; Williams, Justin A.; Stillwell, William; Bittman, Robert; Shaikh, Saame Raza; Wassall, Stephen R.Dietary 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.Item Vitamin E - phosphatidylethanolamine interactions in mixed membranes with sphingomyelin: Studies by 2H NMR(Elsevier, 2020-09) Cavazos, Andres T.; Kinnun, Jacob J.; Williams, Justin A.; Wassall, Stephen R.; Chemistry and Chemical Biology, School of ScienceAmong 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.