Browsing by Subject "Cell membranes"

Now showing 1 - 7 of 7
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    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.
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    A biophysical study of the antibiotic beauvericin
    (1978) Braden, Bradford Carl
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    The Effect of Acyl Chain Unsaturation on Phospholipid Bilayer
    (2010-02-26T17:51:02Z) Soni, Smita Pravin; Wassall, Stephen R.; Petrache, Horia; Kemple, Marvin D.; Rader, Andrew J.
    Each biological cell is surrounded by a membrane that consists of many different kinds of lipids. The lipids are mainly composed of phospholipids, which form a fluid bilayer that serves as the platform for the function of membrane bound proteins regulating cellular activity. In the research described in this thesis we employed solid state 2H NMR, complemented by DSC (differential scanning calorimetry) and MD (molecular dynamics) simulations, to study the effect of PUFA (polyunsaturated fatty acids) and TFA (trans fatty acids) on molecular organization in protein-free model membranes of controlled composition. These two classes of unsaturated fatty acid incorporate into membrane lipids and have, respectively, a beneficial and harmful impact on health. The aim is to gain insight into the molecular origin of this behavior. DHA (docosahexaenoic acid), which with 6 "natural" cis double bonds is the most highly unsaturated PUFA found in fish oils, and EA (elaidic acid), which with only a single "unnatural" trans double bond is the simplest manmade TFA often found in commercially produced food, were the focus. 2H NMR spectra for [2H31]-N-palmitoylsphingomyelin ([2H31]16:0SM) in SM/16:0-22:6PE (1-palmitoyl-2-docosahexaenoylphosphatidylethanolamine)/cholesterol (1:1:1 mol) mixed membranes were recorded. This system served as our PUFA-containing model. The spectra are consistent with lateral separation into nano-sized (< 20 nm) domains that are SM-rich/cholesterol-rich (raft), characterized by higher chain order, and DHA-rich/cholesterol-poor (non-raft), characterized by lower chain order. The aversion cholesterol has for DHA, as opposed to the affinity cholesterol has for predominantly saturated SM, excludes the sterol from DHA-containing PE-rich domains and DHA from SM-rich/cholesterol-rich domains. It is the formation of highly disordered membrane domains that we hypothesize is responsible, in part, for the diverse health benefits associated with dietary consumption of DHA. 2H NMR spectra for 1-elaidoyl-2-[2H35]stearoylphosphatidylcholine (t18:1-[2H35]18:0PC) and 1-oleoyl-2-[2H35]stearoylphosphatidylcholine (c18:1-[2H35]18:0PC) were recorded to compare membranes with respect to a trans vs. cis ("natural") double bond. The spectra indicate that while a trans double bond produces a smaller deviation from linear conformation than a cis double bond, membrane order is decreased by a comparable amount because the energy barrier to rotation about the C-C single bonds either side of a trans or cis double bond is reduced. Although EA adopts a conformation somewhat resembling a saturated fatty acid, the TFA is almost as disordered as its cis counterpart oleic acid (OA). We speculate that EA could be mistaken for a saturated fatty acid and infiltrate lipid rafts to disrupt the high order therein that is necessary for the function of signaling proteins.
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    Increased Resurgent Sodium Currents (INaR) in Inherited and Acquired Disorders of Excitability
    (2012-08-07) Piekarz, Andrew D.; Cummins, Theodore R.; Nicol, Grant D.; Vasko, Michael R.; Hudmon, Andrew; Khanna, Rajesh
    Voltage-gated sodium channels (VGSCs) are dynamic membrane spanning proteins which mediate the rapid influx of Na+ during the upstroke of the action potential (AP). In addition to the large inward Na+ currents responsible for the upstroke of the AP, some VGSC isoforms produce smaller, subthreshold Na+ currents, which can influence the excitable properties of neurons. An example of such a subthreshold current is resurgent Na+ current (INaR). These unusual currents are active during repolarization of the membrane potential, where the channel is normally refractory to activity. INaR exhibit slow gating kinetics and unusual voltage-dependence derived from a novel mechanism of channel inactivation which allows the channel to recover through an open configuration resulting in membrane depolarization early in the falling phase of the AP, ultra-fast re-priming of channels, and multiple AP spikes. Although originally identified in fast spiking central nervous system (CNS) neurons, INaR has recently been observed in a subpopulation of peripheral dorsal root ganglion (DRG) neurons. Because INaR is believed to contribute to spontaneous and high frequency firing of APs, I have hypothesized that increased INaR may contribute to ectopic AP firing associated with inherited and acquired disorders of excitability. Specifically, this dissertation explores the mechanisms which underlie the electrogenesis of INaR in DRG neurons and determines whether the biophysical properties of these unique currents were altered by mutations that cause inherited muscle and neuronal channelopathies or in an experimental model of nerve injury. The results demonstrate that (1) multiple Na+ channel isoforms are capable of producing INaR in DRG neurons, including NaV1.3, NaV1.6, and NaV1.7, (2) inherited muscle and neuronal channelopathIy mutations that slow the rate of channel inactivation increase INaR amplitude, (3) temperature sensitive INaR produced by select skeletal muscle channelopthy mutations may contribute to the triggering of cold-induced myotonia, and (4) INaR amplitude and distribution is significantly increased two weeks post contusive spinal cord injury (SCI). Taken together, results from this dissertation provide foundational knowledge of the properties and mechanism of INaR in DRG neurons and indicates that increased INaR likely contributes to the enhanced membrane excitability associated with multiple inherited and acquired disorders of excitability.
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    Regulating Lipid Organization and Investigating Membrane Protein Properties in Physisorbed Polymer-tethered Membranes
    (2012-08-07) Siegel, Amanda P.; Naumann, Christoph A.; Minto, Robert; Thompson, David H. Pai; Ritchie, Kenneth
    Cell membranes have remarkable properties both at the microscopic level and the molecular level. The current research describes the use of physisorbed polymer-grafted lipids in model membranes to investigate some of these properties on both of these length scales. On the microscopic scale, plasma membranes can be thought of as heterogenous thin films. Cell membranes adhered to elastic substrates are capable of sensing substrate/film mismatches and modulating their membrane stiffness to more closely match the substrate. Membrane/substrate mismatch can be modeled by constructing lipopolymer-enriched lipid monolayers with different bending stiffnesses and physisorbing them to rigid substrates which causes buckling. This report describes the use of atomic force microscopy and epimicroscopy to characterize these buckled structures and to illustrate the use of the buckled structures as diffusion barriers in lipid bilayers. In addition, a series of monolayers with varying bending stiffnesses and thicknesses are constructed on rigid substrates to analyze changes in buckling patterns and relate the experimental results to thin film buckling theory. On the molecular scale, plasma membranes can also be thought of as heterogeneous mixtures of lipids where the specific lipid environment is a crucial factor affecting membrane protein function. Unfortunately, heterogeneities involving cholesterol, labeled lipid rafts, are small and transient in live cells. To address this difficulty, the present work describes a model platform based on polymer-supported lipid bilayers containing stable raft-mimicking domains into which transmembrane proteins are incorporated (αvβ3, and α5β1integrins). This flexible platform enables the use of confocal fluorescence fluctuation spectroscopy to quantitatively probe the effect of cholesterol concentrations and the binding of native ligands (vitronectin and fibronectin for αvβ3, and α5β1) on protein oligomerization state and on domain-specific protein sequestration. In particular, the report shows significant ligand-induced integrin sequestration with a low level of dimerization. Cholesterol concentration increases rate of dimerization, but only moderately. Ligand addition does not affect rate of dimerization in either system. The combined results strongly suggest that ligands induce changes to integrin conformation and/or dynamics without inducing changes in integrin oligomerization state, and in fact these ligand-induce conformational changes impact protein-lipid interactions.