Purification and electron microscopic characterization of the membrane subunit (IICB(Glc)) of the Escherichia coli glucose transporter |
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Authors: | Zhuang J Gutknecht R Flükiger K Hasler L Erni B Engel A |
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Institution: | M. E. Müller-Institute for Microscopic Structural Biology at the Biocenter, University of Basel, Klingelbergstrasse 70, Basel, CH-4056, Switzerland. zhuang@pyl.unibe.ch |
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Abstract: | The glucose transporter of the bacterial phosphotransferase system mediates sugar transport across the cytoplasmic membrane concomitant with sugar phosphorylation. It consists of a cytoplasmic subunit IIA(Glc) and the transmembrane subunit IICB(Glc). IICB(Glc) was purified to homogeneity by urea/alkali washing of membranes and nickel-chelate affinity chromatography. About 1.5 mg highly pure IICB(Glc) representing 77% of the total activity present in the membranes was obtained from 8g (wet weight) of cells. IICB(Glc) was reconstituted into lipid bilayers by temperature-controlled dialysis to yield small 2D crystals and by a rapid detergent-dilution procedure to yield densely packed vesicles. Electron microscopy and digital image processing of the negatively stained 2D crystals revealed a trigonal lattice with a unit cell size of a = b = 14.5 nm. The unit cell morphology exhibited three dimers of IICB(Glc) surrounding the threefold symmetry center. Single particle analysis of IICB(Glc) in proteoliposomes obtained by detergent dialysis also showed predominantly dimeric structures. |
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