Hydrogen atoms are at the limit of visibility in X-ray structures even at high resolution. Neutron macromolecular crystallography (NMX) is an unambiguous method to locate hydrogens and study the significance of hydrogen bonding interactions in biological systems. Since NMX requires very large crystals, very few neutron structures of proteins have been determined yet. In addition, the most common hydrogen isotope ¹H gives rise to significant background due to its large incoherent scattering cross-section. Therefore, it is advantageous to substitute as many hydrogens as possible with the heavier isotope ²H (deuterium) to reduce the sample volume requirement. While the solvent exchangeable hydrogens can be substituted by dissolving the protein in heavy water, complete deuterium labelling – perdeuteration – requires the protein to be expressed in heavy water with a deuterated carbon source. In this work, we developed an optimized method for large scale production of deuterium-labelled bacterial outer membrane protein F (OmpF) for NMX. OmpF was produced using deuterated media with different carbon sources. Mass spectrometry verified the integrity and level of deuteration of purified OmpF. Perdeuterated OmpF crystals diffracted X-rays to a resolution of 1.9 Å. This work lays the foundation for structural studies of membrane protein by neutron diffraction in future.

即使在高分辨率下，氢原子也处于 X 射线结构中的可见度极限。中子大分子晶体学 (NMX) 是一种明确的方法来定位氢并研究生物系统中氢键相互作用的重要性。由于 NMX 需要非常大的晶体，因此很少有蛋白质的中子结构被确定。此外，最常见的氢同位素¹ H 由于其大的非相干散射截面而引起显着的背景。因此，用较重的同位素²取代尽可能多的氢是有利的H（氘）以减少样品体积要求。虽然溶剂可交换氢可以通过将蛋白质溶解在重水中来替代，但完整的氘标记 - 全氘化 - 需要蛋白质在重水中表达，并带有氘化碳源。在这项工作中，我们开发了一种用于大规模生产 NMX 的氘标记细菌外膜蛋白 F (OmpF) 的优化方法。OmpF 是使用具有不同碳源的氘化介质生产的。质谱法验证了纯化 OmpF 的完整性和氘化水平。经氘化的 OmpF 晶体衍射 X 射线，分辨率为 1.9 Å。该工作为今后利用中子衍射研究膜蛋白的结构奠定了基础。