Molecular-scale structural and functional characterization of sparsely tethered bilayer lipid membranes

Surface-tethered biomimetic bilayer membranes (tethered bilayer lipid membranes (tBLMs)) were formed on gold surfaces from phospholipids and a synthetic 1-thiahexa(ethylene oxide) lipid, WC14. They were characterized using electrochemical impedance spectroscopy, neutron reflection (NR), and Fourier-transform infrared reflection-absorption spectroscopy (FT-IRRAS) to obtain functional and structural information. The authors found that electrically insulating membranes (conductance and capacitance as low as 1 microS cm(-2) and 0.6 microF cm(-2), respectively) with high surface coverage (>95% completion of the outer leaflet) can be formed from a range of lipids in a simple two-step process that consists of the formation of a self-assembled monolayer (SAM) and bilayer completion by "rapid solvent exchange." NR provided a molecularly resolved characterization of the interface architecture and, in particular, the constitution of the space between the tBLM and the solid support. In tBLMs based on SAMs of pure WC14, the hexa(ethylene oxide) tether region had low hydration even though FT-IRRAS showed that this region is structurally disordered. However, on mixed SAMs made from the coadsorption of WC14 with a short-chain "backfiller," beta-mercaptoethanol, the submembrane spaces between the tBLM and the substrates contained up to 60% exchangeable solvent by volume, as judged from NR and contrast variation of the solvent. Complete and stable "sparsely tethered" BLMs (stBLMs) can be readily prepared from SAMs chemisorbed from solutions with low WC14 proportions. Phospholipids with unsaturated or saturated, straight or branched chains all formed qualitatively similar stBLMs.This work was supported by the National Science Foundation CBET-0555201 and 0457148. One of the authors M.L. and the AND/R instrument were supported by the National Institutes of Health under Grant No. 1 R01 RR14812 and by the Regents of the University of California

Protein Incorporation in Solid-Supported Model Lipid Membranes

This report was commisioned by NIST Center for Neutron Researc

A thermodynamic study of ketoreductase-catalyzed reactions 3. Reduction of 1-phenyl-1-alkanones in non-aqueous solvents

The equilibrium constants K for the reactions (1-phenyl-1-alkanone + 2-propanol = 1-phenyl-1-alkanol + acetone) in the solvents n-pentane and n-hexane have been determined by using gas chromatography at the temperature 298.15 K. The 1-phenyl-1-alkaones included in this study were: 1-phenyl-1-ethanone, 1-phenyl-1-propanone, 1-phenyl-1-butanone, 1-phenyl-1-pentanone, 1-phenyl-1-hexanone, and 1-phenyl-1-heptanone. The equilibrium constants for the reaction involving 1-phenyl-1-ethanone were measured in the solvent n-hexane as a function of temperature (288 K to 308 K). The calculated thermodynamic quantities for the 1-phenyl-1-ethanone reaction at T = 298.15 K are: K = 0.2177 ± 0.0018; the standard molar Gibbs free energy change, ∆rGmo=(3.78±0.02)kJ·mol-1, the standard molar enthalpy change, ∆rHmo=(4.53±0.87)kJ·mol-1, and the standard molar entropy change, ∆rSmo=(2.5±2.9)J·K-1·mol-1. The equilibrium constants of 1-phenyl-1-alkanone with an odd number of carbons in alkyl side chain are higher than the equilibrium constants of the preceding 1-phenyl-1-alkanone having an even number of carbons in the side chain and follow a zig-zag pattern with increasing carbon number in the alkyl side chain.© Elsevie

Blockade of non-opioid excitatory effects of spinal Dynorphin A at bradykinin receptors

Dynorphin A (Dyn A) is an endogenous opioid peptide that produces neuroinhibitory (antinociceptive) effects via m, d, and k opioid receptors. However, under chronic pain conditions, up-regulated spinal Dyn A can also interact with bradykinin receptors (BRs) to promote hyperalgesia through a neuroexcitatory (pronociceptive) effect.  These excitatory effects cannot be blocked by an opioid antagonist, and thus are non-opioid in nature. Considering the structural dissimilarity between Dyn A and endogenous BR ligands, bradykinin (BK) and kallidin (KD), this interaction could not be predicted, and provided an opportunity to discover a novel potential neuroexcitatory target. Systematic structure-activity relationship (SAR) studies discovered a minimum pharmacophore of Dyn A, [des-Arg7]-Dyn A-(4-11) LYS1044 for antagonist activity at the BRs, along with insights into the key structural features for BRs recognition, i.e., amphipathicity.  The des-Tyr fragment of dynorphin does not bind to opioid receptors.  Intrathecal administration of des-Tyr dynorphin produces hyperalgesia reminiscent of behaviors seen in peripheral neuropathic pain models and at higher doses, neurotoxicity. Our lead ligand LYS1044 blocked Dyn A-(2-13)-induced neuroexcitatory effects in naïve animals and reversed thermal hyperalgesia and mechanical hypersensitivity in a dose-dependent manner in animals with experimental neuropathic pain. Based on these results, ligand LYS1044 might inhibit abnormal pain states by blocking the neuroexcitatory effects of enhanced levels of Dyn A that are seen in experimental models of neuropathic pain and that likely promote excitation mediated by BRs in the spinal cord

Functional Toxin Pores in Tethered Bilayers: Membrane Association of α-hemolysin

This report was commisioned by NIST Center for Neutron Researc

Reconstitution of Cholesterol-Dependent Vaginolysin into Tethered Phospholipid Bilayers: Implications for Bioanalysis

<div><p>Functional reconstitution of the cholesterol-dependent cytolysin vaginolysin (VLY) from <i>Gardnerella vaginalis</i> into artificial tethered bilayer membranes (tBLMs) has been accomplished. The reconstitution of VLY was followed in real-time by electrochemical impedance spectroscopy (EIS). Changes of the EIS parameters of the tBLMs upon exposure to VLY solutions were consistent with the formation of water-filled pores in the membranes. It was found that reconstitution of VLY is a strictly cholesterol-dependent, irreversible process. At a constant cholesterol concentration reconstitution of VLY occurred in a concentration-dependent manner, thus allowing the monitoring of VLY concentration and activity <i>in vitro</i> and opening possibilities for tBLM utilization in bioanalysis. EIS methodology allowed us to detect VLY down to 0.5 nM (28 ng/mL) concentration. Inactivation of VLY by certain amino acid substitutions led to noticeably lesser tBLM damage. Pre-incubation of VLY with the neutralizing monoclonal antibody 9B4 inactivated the VLY membrane damage in a concentration-dependent manner, while the non-neutralizing antibody 21A5 exhibited no effect. These findings demonstrate the biological relevance of the interaction between VLY and the tBLM. The membrane-damaging interaction between VLY and tBLM was observed in the absence of the human CD59 receptor, known to strongly facilitate the hemolytic activity of VLY. Taken together, our study demonstrates the applicability of tBLMs as a bioanalytical platform for the detection of the activity of VLY and possibly other cholesterol-dependent cytolysins.</p> </div

Modification of Tethered Bilayers by Phospholipid Exchange with Vesicles

Phosphatidylcholine and cholesterol exchange between vesicles and planar tethered bilayer lipid membranes (tBLMs) was demonstrated from electrochemical impedance spectroscopy (EIS), fluorescence microscopy (FM), and neutron reflectometry (NR) data. Cholesterol is incorporated into the tBLMs, as determined by the functional reconstitution of the pore forming toxin α-hemolysin (EIS data), attaining cholesterol concentrations nearly equal to that in the donor vesicles. Using fluorescently labeled lipids and cholesterol, FM indicates that the vesicle–tBLM exchange is homogeneous for the lipids but not for cholesterol. NR data with perdeuterated lipids indicates lipid exchange asymmetry with two lipids exchanged in the outer leaflet for every lipid in the inner leaflet. NR and EIS data further show different exchange rates for cholesterol (<i>t</i>_1/2 < 60 min) and phosphatidylcholine (<i>t</i>_1/2 > 4 h). This work lays the foundation for the preparation of robust, lower defect, more biologically relevant tBLMs by essentially combining the two methods of tBLM formation–rapid solvent exchange and vesicle fusion