Protein dynamics at atomic resolution can provide deep insights into the biological activities of proteins and enzymes but they can also make structure and dynamics studies challenging. Despite their well-known biological and pharmaceutical importance, integral membrane protein structure and dynamics studies lag behind those of water-soluble proteins mainly owing to solubility problems that result upon their removal from the membrane. Escherichia coli glycerol facilitator (GF) is a member of the aquaglyceroporin family that allows for the highly selective passive diffusion of its substrate glycerol across the inner membrane of the bacterium. Previous molecular dynamics simulations and hydrogen-deuterium exchange studies suggested that protein dynamics play an important role in the passage of glycerol through the protein pore. With the aim of studying GF dynamics by solution and solid-state nuclear magnetic resonance (NMR) spectroscopy we optimized the expression of isotope-labelled GF and explored various solubilizing agents including detergents, osmolytes, amphipols, random heteropolymers, lipid nanodiscs, bicelles and other buffer additives to optimize the solubility and polydispersity of the protein. The GF protein is most stable and soluble in lauryl maltose neopentyl glycol (LMNG), where it exists in a tetramer-octamer equilibrium. The solution structures of the GF tetramer and octamer were determined by negative-stain transmission electron microscopy (TEM), size-exclusion chromatography small-angle X-ray scattering (SEC-SAXS) and solid-state magic-angle spinning NMR spectroscopy. Although NMR sample preparation still needs optimization for full structure and dynamics studies, negative stain TEM and SEC-SAXS revealed low-resolution structures of the detergent-solubilized tetramer and octamer particles. The non-native octamer appears to form from the association of the cytoplasmic faces of two tetramers, the interaction apparently mediated by their disordered N- and C-termini. This information may be useful in future studies directed at reducing the heterogeneity and self-association of the protein.

中文翻译：

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大肠杆菌甘油促进剂的溶液结构和低聚状态。

原子分辨率下的蛋白质动力学可以提供深入了解蛋白质和酶的生物学活性的信息，但也可能使结构和动力学研究具有挑战性。尽管它们具有众所周知的生物学和药学重要性，但完整的膜蛋白结构和动力学研究仍落后于水溶性蛋白，这主要是由于从膜上去除后的溶解性问题所致。大肠杆菌甘油促进剂（GF）是aquaglyceroporin家族的成员，它允许其底物甘油在细菌内膜上高度选择性地被动扩散。先前的分子动力学模拟和氢-氘交换研究表明，蛋白质动力学在甘油通过蛋白质孔的过程中起着重要作用。为了通过溶液和固态核磁共振（NMR）光谱研究GF动力学，我们优化了同位素标记的GF的表达，并探索了各种增溶剂，包括去污剂，渗透剂，两亲，无规杂聚物，脂质纳米盘，双胞胎和其他缓冲添加剂可优化蛋白质的溶解度和多分散性。GF蛋白最稳定，可溶于十二烷基麦芽糖新戊二醇（LMNG）中，该蛋白以四聚体-八聚体平衡存在。GF四聚体和八聚体的溶液结构通过负染色透射电子显微镜（TEM），尺寸排阻色谱法小角X射线散射（SEC-SAXS）和固态幻角旋转NMR光谱法确定。尽管NMR样品制备仍需要优化以进行完整的结构和动力学研究，但负染色TEM和SEC-SAXS揭示了洗涤剂溶解的四聚体和八聚体颗粒的低分辨率结构。非天然八聚体似乎是由两个四聚体的细胞质表面的结合形成的，相互作用显然是由其无序的N-和C-末端介导的。此信息可能对将来针对减少蛋白质的异质性和自缔合的研究有用。