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    Perdeuteration of cholesterol for neutron scattering applications using recombinant Pichia pastoris

    Moulin, M. and Strohmeier, G.A. and Hirz, M. and Thompson, Katherine C. and Rennie, A.R. and Campbell, R.A. and Pichler, H. and Maric, S. and Forsyth, V.T. and Haertlein, M. (2018) Perdeuteration of cholesterol for neutron scattering applications using recombinant Pichia pastoris. Chemistry and Physics of Lipids 212 , pp. 80-87. ISSN 0009-3084.

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    Abstract

    Deuteration of biomolecules has a great impact on both quality and scope of neutron scattering experiments. Cholesterol is a major component of mammalian cells, where it plays a critical role in membrane permeability, rigidity and dynamics, and contributes to specific membrane structures such as lipid rafts. Cholesterol is the main cargo in low and high-density lipoprotein complexes (i.e. LDL, HDL) and is directly implicated in several pathogenic conditions such as coronary artery disease which leads to 17 million deaths annually. Neutron scattering studies on membranes or lipid-protein complexes exploiting contrast variation have been limited by the lack of availability of fully deuterated biomolecules and especially perdeuterated cholesterol. The availability of perdeuterated cholesterol provides a unique way of probing the structural and dynamical properties of the lipoprotein complexes that underly many of these disease conditions. Here we describe a procedure for in vivo production of perdeuterated recombinant cholesterol in lipid-engineered Pichia pastoris. Using flask and fed-batch fermenter cultures in deuterated minimal medium perdeuteration of the purified cholesterol was verified by mass spectrometry and its use in a neutron scattering study was demonstrated using neutron reflectometry.

    Metadata

    Item Type: Article
    Keyword(s) / Subject(s): Perdeuteration, cholesterol, Pichia pastoris, neutron scattering, lipid engineering
    School: Birkbeck Schools and Departments > School of Science > Biological Sciences
    Depositing User: Katherine Thompson
    Date Deposited: 05 Feb 2018 08:01
    Last Modified: 17 Oct 2019 04:08
    URI: http://eprints.bbk.ac.uk/id/eprint/21096

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