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具有重大影响。胆固醇是哺乳动物细胞的主要成分，在膜渗透性，刚性和动力学方面起着至关重要的作用，并有助于特定的膜结构，例如脂质筏。胆固醇是低密度脂蛋白复合物和高密度脂蛋白复合物（例如LDL，HDL）的主要货物，直接与多种致病性疾病如冠状动脉疾病有关，每年导致1700万人死亡。由于缺乏充分氘化的生物分子，尤其是氘代的胆固醇，利用对比度变化对膜或脂质-蛋白质复合物进行中子散射研究受到了限制。氘代胆固醇的可利用性提供了一种独特的方法来探测许多这些疾病条件下的脂蛋白复合物的结构和动力学性质。在这里，我们描述了在氘化的基本培养基中使用烧瓶和补料分批发酵罐培养物，在脂质工程化的毕赤酵母中体内生产全氘化重组胆固醇。纯化的胆固醇的氘化已通过质谱法验证，其在中子散射研究中的应用已通过ILL的FIGARO仪器的中子反射法测量得到了证明。