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Three‐dimensional crystallization of the Escherichia coli glycerol‐3‐phosphate transporter: A member of the major facilitator superfamily
Author(s) -
Lemieux M. Joanne,
Song Jinmei,
Kim Myong Jin,
Huang Yafei,
Villa Anthony,
Auer Manfred,
Li XiaoDan,
Wang DaNeng
Publication year - 2003
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.03276603
Subject(s) - crystallization , crystallography , escherichia coli , chemistry , biology , biochemistry , organic chemistry , gene
Here we report the successful three‐dimensional crystallization of GlpT, the glycerol‐3‐phosphate transporter from Escherichia coli inner membrane. GlpT possesses 12 transmembrane α‐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 Å 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 Å resolution and were suitable for structure determination by X‐ray crystallography.