Browsing by Author "Wacklin, HP"

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    Changes in molecular composition and packing during lipid membrane reconstitution from phospholipid-surfactant micelles
    (Royal Society of Chemistry, 2009-02-07) Lee, CS; Wacklin, HP; Bain, CD
    Total internal reflection (TIR) Raman spectroscopy was used to investigate changes in the properties of phospholipid bilayers at the quartz-water interface during the process of membrane reconstitution from lipid-detergent micelles. The non-ionic detergent beta-D-dodecyl maltoside (beta-DDM) was used in 9 : 1 ratio to deuterated and non-deuterated dipalmitoylphosphatidyl choline (DPPC and d(75)-DPPC) to form detergent-lipid micelles. Exposure of the quartz surface to decreasing concentrations of such detergent-lipid solutions resulted in the formation of a d(75)-DPPC bilayer indistinguishable from that of a bilayer formed by fusion of small unilamellar vesicles of the pure lipid, indicating that the use of detergent micelles does not alter the packing or conformation of lipids, or leave a residue of surfactant in the bilayer. Polarised measurements of d(75)DPPC spectra in the C-H and C-D regions at each step of the process showed that the lipid surface density increases as the detergent is removed by rinsing and replaced by more lipid. The absence of shifts in the frequencies demonstrates that DPPC is in a gel-like conformation even at high relative surfactant fractions. The TIR geometry also allowed us to record high resolution spectra in the low frequency C-D bending region of d(75)-DPPC, which showed that the lipid molecules have an upright orientation with respect to the support surface. © 2009, Royal Society of Chemistry
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    Composition and Asymmetry in Supported Membranes Formed by Vesicle Fusion
    (Royal Society of Chemistry, 2011-06-21) Wacklin, HP
    The structure and formation of supported membranes at silica surfaces by vesicle fusion was investigated by neutron reflectivity and quartz crystal microbalance (QCM-D) measurements. The structure of equimolar phospholipid mixtures of DLPC?DPPC, DMPC?DPPC, and DOPC?DPPC depends intricately on the vesicle deposition conditions. The supported bilayer membranes exhibit varying degrees of compositional asymmetry between the monolayer leaflets, which can be modified by the deposition temperature as well as the salt concentration of the vesicle solution. The total lipid composition of the supported bilayers differs from the composition of the vesicles in solution, and the monolayer proximal to the silica surface is always enriched in DPPC compared to the distal monolayer. The results, which show unambiguougsly that some exchange and rearrangement of lipids occur during vesicle deposition, can be rationalized by considering the effects of salt screening and temperature on the rates of lipid exchange, rearrangement, and vesicle adsorption, but there is also an intricate dependence on the lipid?lipid interactions. Thus, although both symmetric and asymmetric supported bilayers can be prepared from vesicles, the optimal conditions are sensitive to the lipid composition of the system. © 2011, Royal Society of Chemistry
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    Cyclotides insert into lipid bilayers to form membrane pores and destabilize the membrane through hydrophobic and phosphoethanolamine-specific interactions
    (American Society for Biochemistry and Molecular Biology, 2012-12-21) Wang, CK; Wacklin, HP; Craik, DJ
    Cyclotides are a family of plant-derived circular proteins with potential therapeutic applications arising from their remarkable stability, broad sequence diversity, and range of bioactivities. Their membrane-binding activity is believed to be a critical component of their mechanism of action. Using isothermal titration calorimetry, we studied the binding of the prototypical cyclotides kalata B1 and kalata B2 (and various mutants) to dodecylphosphocholine micelles and phosphoethanolamine-containing lipid bilayers. Although binding is predominantly an entropy-driven process, suggesting that hydrophobic forces contribute significantly to cyclotide-lipid complex formation, specific binding to the phosphoethanolamine-lipid headgroup is also required, which is evident from the enthalpic changes in the free energy of binding. In addition, using a combination of dissipative quartz crystal microbalance measurements and neutron reflectometry, we elucidated the process by which cyclotides interact with bilayer membranes. Initially, a small number of cyclotides bind to the membrane surface and then insert first into the outer membrane leaflet followed by penetration through the membrane and pore formation. At higher concentrations of cyclotides, destabilization of membranes occurs. Our results provide significant mechanistic insight into how cyclotides exert their bioactivities. © 2012, American Society for Biochemistry and Molecular Biology.
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    Interfacial mechanism of phospholipase A(2): pH-dependent inhibition and Me-β-cyclodextrin activation.
    (American Chemical Society, 2009-06-30) Wacklin, HP
    The pH-dependent activity of phospholipase A(2) (PLA(2)) from Naja mossambica mossambica venom and the membrane-water partitioning of the lipid hydrolysis products were investigated in solid-supported palmitoyl-oleyl-phosphatidylcholine-d(31) (POPC-d(31)) membranes using neutron reflection. At pH 5, PLA(2) interacts only weakly with the substrate membrane and leads to no observable membrane breakdown, which is consistent with protonation of the catalytic histidine (His48, pK(a) similar to 6.2). The rate of the lyso-lipid partitioning into the solution phase is the same at pH 9 as at pH 7.4, and the relative membrane-water partitioning of the products is essentially the same; that is, the fatty acid accumulates in the membrane, and only the lyso-lipid is solubilized. However, Me-β-cyclodextrin (Me-β-CD) activates PLA(2) irrespective of pH by facilitating the solubilization of the lyso-lipid product, but not the fatty acid, of which only 22% is encapsulated at pH 9. Since no product solubilization is observed at pH 5 in the absence of Me-β-CD, this suggests that the hydrolytic mechanism of PLA(2) is not fully disabled at pH 5 but is inhibited by a mechanism, which is counteracted by Me-p-M-mediated release of the lyso-lipid. Me-β-CD does not interact with the substrate membrane, which indicates that at low pH the product extraction occurs directly from the enzyme active site outside the immediate membrane-water interface, whereas at pH 7-9, direct solubilization of the lyso-lipid from the membrane can also contribute to activation of PLA(2). © 2009, American Chemical Society
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    Synthesis of perdeuterated 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine ([D82]POPC) for neutron studies on bilayer lipid membranes
    (International Conference on Neutron Scattering, 2017-07-12) Yepuri, NR; Darwish, TA; Leung, A; Krause-Heuer, AM; Wacklin, HP; Delhom, R; Holden, PJ
    The complexity of the chemical synthesis of completely deuterium-labelled unsaturated lipids has meant that most neutron experiments, to date, have been restricted to saturated phospholipid species, the behaviour of which under physiological conditions may not be representative of the unsaturated varieties found in biological membranes. Unsaturated lipids occur widely in nature and are crucial for the fluidity of cell membranes. Biologically relevant phospholipids such as 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) are typically asymmetric and cis-9 unsaturated in the sn-2-acyl chain, where as the sn-1 chain is often saturated. Commercially available sn-1 chain deuterated [D31]1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine([D31]POPC) does not provide enough contrast for detailed structural investigation through neutron studies, and is nearly perfectly contrast-matched to most proteins and membrane binding peptides in heavy water, which makes them neutron invisible. With this background in mind, we have developed a method for the synthesis of perdeuterated POPC and its partially labelled form.[1] The structure of a supported bilayer membrane formed from these lipids was determined by measuring neutron reflectivity in a series of solvent contrasts, which will be discussed in this paper.