Institut Laue-Langevin, 71, Avenue des Martyrs, Grenoble 38042, France; Faculty of Natural Sciences, Keele University, Keele, Staffordshire ST5 5BG, United Kingdom.
acib, Austrian Centre of Industrial Biotechnology GmbH, 8010 Graz, Austria; Institute of Organic Chemistry, NAWI Graz, Graz University of Technology, 8010 Graz, Austria.
Chem Phys Lipids. 2018 May;212:80-87. doi: 10.1016/j.chemphyslip.2018.01.006. Epub 2018 Jan 31.
Deuteration of biomolecules has a major 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 by neutron reflectometry measurements using the FIGARO instrument at the ILL.
生物分子的氘化对中子散射实验的质量和范围都有重大影响。胆固醇是哺乳动物细胞的主要成分，在膜通透性、刚性和动力学方面起着关键作用，并有助于形成特定的膜结构，如脂筏。胆固醇是低密和高密脂蛋白复合物（即 LDL、HDL）的主要成分，并直接涉及到几种致病情况，如每年导致 1700 万人死亡的冠心病。利用对比变化进行膜或脂质-蛋白质复合物的中子散射研究受到缺乏完全氘化生物分子，尤其是全氘化胆固醇的限制。全氘化胆固醇的可用性为研究许多此类疾病条件下的脂蛋白复合物的结构和动力学特性提供了一种独特的方法。在这里，我们描述了一种在使用氘化最小培养基的摇瓶和分批发酵罐培养物中，在脂质工程化毕赤酵母中体内生产全氘化重组胆固醇的方法。通过质谱法验证了纯化胆固醇的氘化程度，并通过在 ILL 的 FIGARO 仪器上使用中子反射计测量证明了其在中子散射研究中的用途。
