Three-dimensional crystallization of the Escherichia coli glycerol-3-phosphate transporter: a member of the major facilitator superfamily.
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.
Lemieux
M Joanne
MJ
Skirball Institute of Biomolecular Medicine and Department of Cell Biology, New York University School of Medicine, New York, New York 10016, USA.
Song
Jinmei
J
Kim
Myong Jin
MJ
Huang
Yafei
Y
Villa
Anthony
A
Auer
Manfred
M
Li
Xiao-Dan
XD
Wang
Da-Neng
DN
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
D003902:Detergents; C488962:GlpT protein, E coli; D026901:Membrane Transport Proteins; D010743:Phospholipids; D011993:Recombinant Fusion Proteins