Three-dimensional crystallization of the Escherichia coli glycerol-3-phosphate transporter:: A member of the major facilitator superfamily
Three-dimensional crystallization of the Escherichia coli glycerol-3-phosphate transporter:: A member of the major facilitator superfamily
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DOI：
10.1110/ps.03276603
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发表时间：
2003-12-01
期刊：
影响因子：
8
通讯作者：
Wang, DN
中科院分区：
文献类型：
--
作者：
Lemieux, MJ;Song, JM;Wang, DN
在这里，我们报告了成功的三维结晶的GlpT，从大肠杆菌内膜的甘油-3-磷酸转运蛋白。GlpT具有12个跨膜α螺旋，是主要易化剂超家族的成员。它介导甘油-3-磷酸与无机磷酸盐的跨膜交换。大约20个磷脂分子每蛋白质，确定为带负电荷的磷脂酰乙醇胺，磷脂酰甘油，和心磷脂，所需的纯化GlpT的单分散性。分析尺寸排阻色谱法被证明是有效的，在确定洗涤剂的GlpT单分散性。九个这样的洗涤剂后来用于GlpT结晶。筛选晶体成核进行了各种聚乙二醇作为沉淀剂在一个很宽的pH范围。随后通过有限的蛋白质水解和质谱鉴定刚性蛋白质核心，导致更有序的晶体。这些晶体在二维中表现出3.7埃分辨率的顺序。然而，在第三维中的堆叠是部分无序的。通过使用洗涤剂混合物和操纵结晶溶液中的离子相互作用，克服了这种堆积问题。得到的GlpT晶体各向同性衍射至3.3埃分辨率，并且适合于通过X射线晶体学进行结构测定。
Here we report the successful three-dimensional crystallization of GlpT, the glycerol-3-phosphate transporter from Escherichia coli inner membrane. GlpT possesses 12 transmembrane alpha-helices and is a member of the major facilitator superfamily. It mediates the exchange of glycerol-3-phosphate for inorganic phosphate across the membrane. Approximately 20 phospholipid molecules per protein, identified as negatively charged phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin, were required for the monodispersity of purified GlpT. Analytical size-exclusion chromatography proved to be efficient in identifying detergents for GlpT monodispersity. Nine such detergents were later used for GlpT crystallization. Screening for crystal nucleation was carried out with a variety of polyethylene glycols as the precipitant over a wide pH range. Subsequent identification of a rigid protein core by limited proteolysis and mass spectroscopy resulted in better-ordered crystals. These crystals exhibited order to 3.7 Angstrom resolution in two dimensions. However, the stacking in the third dimension was partially disordered. This stacking problem was overcome by using a detergent mixture and manipulating the ionic interactions in the crystallization solution. The resulting GlpT crystals diffracted isotropically to 3.3 Angstrom resolution and were suitable for structure determination by X-ray crystallography.