title: Three-dimensional crystallization of the Escherichia coli glycerol-3-phosphate transporter: a member of the major facilitator superfamily.
abstract: 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 A 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 A resolution and were suitable for structure determination by X-ray crystallography.
authors: Skirball Institute of Biomolecular Medicine and Department of Cell Biology, New York University School of Medicine, New York, New York 10016, USA.
authors: M Joanne
authors: MJ
authors: Lemieux
authors: Jinmei
authors: J
authors: Song
authors: Myong Jin
authors: MJ
authors: Kim
authors: Yafei
authors: Y
authors: Huang
authors: Anthony
authors: A
authors: Villa
authors: Manfred
authors: M
authors: Auer
authors: Xiao-Dan
authors: XD
authors: Li
authors: Da-Neng
authors: DN
authors: Wang
mesh_terms: D002846:Chromatography, Affinity; D003460:Crystallization; D018360:Crystallography, X-Ray; D003902:Detergents; D004926:Escherichia coli / Q000737:chemistry*; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D003902:Detergents; C488962:GlpT protein, E coli; D026901:Membrane Transport Proteins; D010743:Phospholipids; D011993:Recombinant Fusion Proteins