Browsing by Author "Hossain, KR"

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    Stereoselective synthesis of perdeuterated phytanic acid, its phospholipid derivatives and their formation into lipid model membranes for neutron reflectivity studies
    (Elsevier, 2014-10-01) Yepuri, NR; Holt, SA; Moraes, G; Holden, PJ; Hossain, KR; Valenzuela, SM; Darwish, TA; James, M
    We describe a straightforward method, for synthesis of large scale (gram quantities) of highly deuterated phytanic acid from commercially available phytol while preserving the stereochemistry around the chiral centres. The subsequent synthesis of tail-deuterated analogues of the archeabacterial membrane lipids 1,2-di(3RS,7R,11R-phytanyl)-sn-glycero-3-phosphocholine (DPEPC) and 1,2-di(3RS,7R,11R-phytanoyl)-sn-glycero-3-phosphocholine (DPhyPC) from perdeuterated phytanic acid is also described. Both lipids were employed in construction of two different model membranes, namely Langmuir monolayers and a tethered bilayer membrane (TBM) on a solid substrate, characterised by pressure area isotherm and neutron reflectometry techniques. At 10 mN/m pressure the head-group thickness of both monolayers was similar while the thickness of the tail region was significantly larger for tail-deuterated DPhyPC, which was evident from a smaller area per molecule. At 20 mN/m the thickness of the head and tail regions in both lipids was comparable, yet the area per molecule of tail-deuterated DPhyPC was 10% smaller than tail-deuterated DPEPC. In the TBM bilayer model membrane, the thickness of the lipid tails in both inner and outer leaflets was 8.2 Å, giving a total of 16.4 Å. Deuteration enabled unambiguous determination of the relative proportion of the hydrogenous tether, phospholipid and subphase. © 2014, Elsevier Ireland Ltd.
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    X-ray and neutron reflectivity study of the membrane-bound CLIC1 protein at the air-water interface
    (Australian Institute of Nuclear Science and Engineering, 2016-11-29) Hossain, KR; Holt, SA; Khamici, HA; Valenzuela, SM
    The CLIC proteins are a ubiquitous family of Chloride Intracellular Ion Channel proteins that are evolutionarily conserved across species and exist as a soluble form and an integral membrane-bound form 1. The X-ray structure of the soluble form of a number of CLIC proteins has been solved and their putative transmembrane domain identified 2-5. However, the factors which facilitate the membrane insertion, the structural transition between these two forms and the structural features of the membrane-bound form for this class of proteins remain largely unknown. In an attempt to answer these questions, we have employed biophysical techniques to study the interaction of wild-type and mutant versions of the protein CLIC1 with monolayers prepared using various mixtures of different phospholipids and sterol molecules. Our findings have demonstrated that cholesterol plays a crucial role for the penetration of CLIC1 into the hydrophobic tails of the lipid monolayer with the protein occupying an area per molecule between 5-7 nm2. We have also demonstrated for the first time that CLIC1 interaction with cholesterol is dependent on the intact 3β-OH group in the sterol ring and that the GXXXG motif in CLIC1 acts as the cholesterol-binding site used by the protein for its initial recognition and binding to membrane cholesterol.
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    X-ray and neutron reflectivity study shows that CLIC1 undergoes cholesterol-dependent structural reorganization in lipid monolayers
    (ACS Publications, 2017-10-17) Hossain, KR; Holt, SA; Le Brun, AP; Al Khamici, H; Valenzuela, SM
    CLIC1 belongs to the ubiquitous family of chloride intracellular ion channel proteins that are evolutionarily conserved across species. The CLICs are unusual in that they exist mainly as soluble proteins but possess the intriguing property of spontaneous conversion from the soluble to an integral membrane-bound form. This conversion is regulated by the membrane lipid composition, especially by cholesterol, together with external factors such as oxidation and pH. However, the precise physiological mechanism regulating CLIC1 membrane insertion is currently unknown. In this study, X-ray and neutron reflectivity experiments were performed to study the interaction of CLIC1 with different phospholipid monolayers prepared using POPC, POPE, or POPS with and without cholesterol in order to better understand the regulatory role of cholesterol in CLIC1 membrane insertion. Our findings demonstrate for the first time two different structural orientations of CLIC1 within phospholipid monolayers, dependent upon the absence or presence of cholesterol. In phospholipid monolayers devoid of cholesterol, CLIC1 was unable to insert into the lipid acyl chain region. However, in the presence of cholesterol, CLIC1 showed significant insertion within the phospholipid acyl chains occupying an area per protein molecule of 6−7 nm2 with a total CLIC1 thickness ranging from ∼50 to 56 Å across the entire monolayer. Our data strongly suggests that cholesterol not only facilitates the initial docking or binding of CLIC1 to the membrane but also promotes deeper penetration of CLIC1 into the hydrophobic tails of the lipid monolayer. © 2017 American Chemical Society