Wadsten Pia, Wöhri Annemarie B, Snijder Arjan, Katona Gergely, Gardiner Alastair T, Cogdell Richard J, Neutze Richard, Engström Sven
Department of Chemical and Biological Engineering, Pharmaceutical Technology, Chalmers University of Technology, Göteborg, Sweden.
J Mol Biol. 2006 Nov 17;364(1):44-53. doi: 10.1016/j.jmb.2006.06.043. Epub 2006 Jul 7.
Bicontinuous lipidic cubic phases can be used as a host for growing crystals of membrane proteins. Since the cubic phase is stiff, handling is difficult and time-consuming. Moreover, the conventional cubic phase may interfere with the hydrophilic domains of membrane proteins due to the limited size of the aqueous pores. Here, we introduce a new crystallization method that makes use of a liquid analogue of the cubic phase, the sponge phase. This phase facilitates a considerable increase in the allowed size of aqueous domains of membrane proteins, and is easily generalised to a conventional vapour diffusion crystallisation experiment, including the use of nanoliter drop crystallization robots. The appearance of the sponge phase was confirmed by visual inspection, small-angle X-ray scattering and NMR spectroscopy. Crystals of the reaction centre from Rhodobacter sphaeroides were obtained by a conventional hanging-drop experiment, were harvested directly without the addition of lipase or cryoprotectant, and the structure was refined to 2.2 Angstroms resolution. In contrast to our earlier lipidic cubic phase reaction centre structure, the mobile ubiquinone could be built and refined. The practical advantages of the sponge phase make it a potent tool for crystallization of membrane proteins.
双连续脂质立方相可作为生长膜蛋白晶体的宿主。由于立方相坚硬，处理起来困难且耗时。此外，由于水孔尺寸有限，传统的立方相可能会干扰膜蛋白的亲水区。在此，我们介绍一种新的结晶方法，该方法利用立方相的液体类似物——海绵相。这个相有助于显著增加膜蛋白水相区域的允许尺寸，并且很容易推广到传统的气相扩散结晶实验，包括使用纳升级液滴结晶机器人。通过目视检查、小角X射线散射和核磁共振光谱证实了海绵相的出现。球形红杆菌反应中心的晶体通过传统的悬滴实验获得，直接收获，无需添加脂肪酶或冷冻保护剂，并且结构精修至2.2埃分辨率。与我们早期的脂质立方相反应中心结构相比，可构建并精修移动泛醌。海绵相的实际优势使其成为膜蛋白结晶的有力工具。
