van Dalsen Leendert, Weichert Dietmar, Caffrey Martin
Membrane Structural and Functional Biology Group, School of Medicine and School of Biochemistry and Immunology, Trinity College Dublin, Dublin D02 R590, Ireland.
J Appl Crystallogr. 2020 Mar 25;53(Pt 2):530-535. doi: 10.1107/S1600576720002289. eCollection 2020 Apr 1.
Digitonin has long been used as a mild detergent for extracting proteins from membranes for structure and function studies. As supplied commercially, digitonin is inhomogeneous and requires lengthy pre-treatment for reliable downstream use. Glyco-diosgenin (GDN) is a recently introduced synthetic surfactant with features that mimic digitonin. It is available in homogeneously pure form. GDN is proving to be a useful detergent, particularly in the area of single-particle cryo-electron microscopic studies of membrane integral proteins. With a view to using it as a detergent for crystallization trials by the or lipid cubic phase method, it was important to establish the carrying capacity of the cubic mesophase for GDN. This was quantified in the current study using small-angle X-ray scattering for mesophase identification and phase microstructure characterization as a function of temperature and GDN concentration. The data show that the lipid cubic phase formed by hydrated monoolein tolerates GDN to concentrations orders of magnitude in excess of those used for membrane protein studies. Thus, having GDN in a typical membrane protein preparation should not deter use of the method for crystallogenesis.
洋地黄皂苷长期以来一直被用作一种温和的去污剂，用于从膜中提取蛋白质以进行结构和功能研究。市售的洋地黄皂苷是不均匀的，需要进行长时间的预处理才能可靠地用于下游实验。糖基薯蓣皂苷元（GDN）是一种最近引入的合成表面活性剂，具有类似洋地黄皂苷的特性。它以均匀纯净的形式提供。事实证明，GDN是一种有用的去污剂，特别是在膜整合蛋白的单颗粒冷冻电子显微镜研究领域。为了将其用作通过 或脂质立方相法进行结晶试验的去污剂，确定立方中间相对GDN的承载能力很重要。在本研究中，使用小角X射线散射对中间相进行鉴定，并将相微观结构表征为温度和GDN浓度的函数，从而对其进行了量化。数据表明，由水合单油酸甘油酯形成的脂质立方相对GDN的耐受性超过了用于膜蛋白研究的浓度几个数量级。因此，在典型的膜蛋白制备中含有GDN不应妨碍使用 方法进行晶体生成。
