Crystalline arrays of the Escherichia coli sn-glycerol-3-phosphate acyltransferase, an integral membrane protein.
The gene encoding the Escherichia coli sn-glycerol-3-phosphate acyltransferase, plsB, was inserted into hybrid plasmids under transcriptional control of the lambda PL and tac promoters. Enzymatic activities 35-50-fold above wild type and a large increase in glycerol-P acyltransferase polypeptide were obtained. Thin section electron microscopy of the cells overproducing the glycerol-P acyltransferase revealed 235-245-A diameter tubular structures associated with the cytoplasmic membrane. These structures were released from the cell by osmotic lysis and purified on Matrex Gel Green A. Subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the dominant protein constituent of the tubules was the glycerol-P acyltransferase. Analysis of tubule-enriched fractions isolated by differential centrifugation revealed a decreased phospholipid to protein ratio as compared to total and cytoplasmic membrane fractions. At high magnification, negative stained tubules displayed ordered arrays of stain-excluding components projecting 50-60 A from the cytoplasmic surface. Optical diffraction patterns from the micrographs contained intense layer lines at (1/78 A) and (1/39 A) along the tubule axis and a prominent spot at (1/62 A) near the equator. From compositional and structural data, 18-37% of the polypeptide volume is estimated to lie within the hydrophobic domain of the tubule membrane.
D000217:Acyltransferases / Q000096:biosynthesis; D003460:Crystallization; D004926:Escherichia coli / Q000201:enzymology*; D005992:Glycerol-3-Phosphate O-Acyltransferase / Q000096:biosynthesis; D008565:Membrane Proteins / Q000096:biosynthesis; D008854:Microscopy, Electron; D010957:Plasmids; D014961:X-Ray Diffraction
D008565:Membrane Proteins; D000217:Acyltransferases; D005992:Glycerol-3-Phosphate O-Acyltransferase