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1.
Figure 4

Figure 4. From: Characterization of the highly conserved VFMGD motif in a bacterial polyisoprenyl-phosphate N-acetylaminosugar-1-phosphate transferase.

The WecAD217N protein mediates increased Und-P-P-GlcNAc production when compared with parental WecA. A: TLC of lipid extractions from MV501 total membranes containing parental WecA (WT) and the D217N mutant WecA total proteins that were incubated with 14C-UDP-GlcNAc. B: Western blot of parental WecA (WT) and D217N mutant proteins using anti-FLAG antibodies. C: Graph depicting the ratio of Und-P-P-GlcNAc product formed per WecA-FLAG protein.

Sarah E Furlong, et al. Protein Sci. 2012 September;21(9):1366-1375.
2.
Figure 6

Figure 6. From: Characterization of the highly conserved VFMGD motif in a bacterial polyisoprenyl-phosphate N-acetylaminosugar-1-phosphate transferase.

Tunicamycin binding assay using parental WecA and D217 replacement mutants. About 40 μg of total membranes containing the various D217 mutant proteins were incubated with a concentration of tunicamycin that inhibits 100% activity of 20 μg of total membranes containing wild-type WecA. The residual tunicamycin, unbound by the mutant proteins, was determined by adding the supernatants to 20 μg of total membranes containing wild-type WecA. The data were represented as a percentage of WecA-FLAG-binding tunicamycin activity. Data represent duplicates from two independent experiments.

Sarah E Furlong, et al. Protein Sci. 2012 September;21(9):1366-1375.
3.
Figure 5

Figure 5. From: Characterization of the highly conserved VFMGD motif in a bacterial polyisoprenyl-phosphate N-acetylaminosugar-1-phosphate transferase.

GlcNAc-1-P transferase activity of the parental WecA (A) and the D217N mutant (B). About 40 μg of total membranes were incubated with increasing amounts of 14C-UDP-GlcNAc for 30 min at 37°C. The lipid fraction was extracted, and then the counts were quantified in a scintillation counter. The enzyme units are expressed as picomoles of UDP-GlcNAc incorporated per milligram of protein. The experiment was performed in triplicate.

Sarah E Furlong, et al. Protein Sci. 2012 September;21(9):1366-1375.
4.
Figure 7

Figure 7. From: Characterization of the highly conserved VFMGD motif in a bacterial polyisoprenyl-phosphate N-acetylaminosugar-1-phosphate transferase.

In vivo and in vitro complementation of alanine replacement mutants in the VFMGD motif. A: Complementation of O7 LPS in E. coli MV501. B: GlcNAc-1-P transferase activity of parental WecA and alanine replacement mutant proteins. Enzyme units are expressed as picomoles of UDP-GlcNAc incorporated per milligram of protein. Experiments were performed in triplicate. C: Expression of the mutant proteins in total membranes as detected by Western blotting using anti-FLAG antibodies.

Sarah E Furlong, et al. Protein Sci. 2012 September;21(9):1366-1375.
5.
Figure 2

Figure 2. From: Characterization of the highly conserved VFMGD motif in a bacterial polyisoprenyl-phosphate N-acetylaminosugar-1-phosphate transferase.

Sulfhydryl labeling accessibility of the cysteine replacement in WecAD217C. The top panel shows the results obtained with biotin maleimide labeling. The second and fourth panels show Western blots with anti-FLAG antibodies. The third panel shows the results of biotin maleimide labeling with MTSET-pretreated samples. WecA was purified by Co2+ affinity chromatography and separated by 14% SDS-PAGE. After transfer to a nitrocellulose membrane, biotin maleimide labeling was detected by a streptavidin-conjugated fluorophore (green signal), and WecA-FLAG proteins were detected using anti-FLAG antibodies (red signal). Biotinylated WecA-FLAG proteins are observed by the merged images, which appear yellow using Odyssey LI-COR Scanner. The cysteine replacement of D217 in the cysteine-less WecA-FLAG protein is labeled with biotin maleimide irrespective of MTSET pretreatment, suggesting that this residue faces the cytosol.

Sarah E Furlong, et al. Protein Sci. 2012 September;21(9):1366-1375.
6.
Figure 3

Figure 3. From: Characterization of the highly conserved VFMGD motif in a bacterial polyisoprenyl-phosphate N-acetylaminosugar-1-phosphate transferase.

In vivo and in vitro complementation of the D217 replacement mutants. A: Complementation of O7 LPS expression in E. coli MV501 by plasmids encoding parental WecA (WT) and various D217 replacement mutants, as determined with silver-stained polyacrylamide gels. The pBAD24 cloning vector was used as a negative control. B: GlcNAc-1-P transferase activity assays performed with total membrane extracts prepared from E. coli MV501 cells carrying pBAD24 or plasmids encoding parental WecA (WT) and D217 replacement mutants. Bars represent means and standard errors of triplicate experiments. C: Expression of the WecA proteins in the membrane extracts used for enzymatic assays, as detected by Western blotting using anti-FLAG antibodies.

Sarah E Furlong, et al. Protein Sci. 2012 September;21(9):1366-1375.
7.
Figure 1

Figure 1. From: Characterization of the highly conserved VFMGD motif in a bacterial polyisoprenyl-phosphate N-acetylaminosugar-1-phosphate transferase.

Topological model of the E. coli WecA. A: Refined topological model of the E. coli WecA. This model was previously established by a combination of bioinformatics, substituted cysteine accessibility experiments, and reporter gene fusions.20 A dotted ellipse indicates the highly conserved V213FMGD217 motif, and its topology has been determined in this work (see Results section). The highly conserved residues that are important for function based on previous research20, 23, 24 are circled. Shading indicates D217. The dotted circle indicates the residue S220. Squares show the control residues for cysteine scanning accessibility experiments used in this work, G181 and S362. B: ClustalW alignment of PNPT members shows the highly conserved VFMGD motif. Alignment shows protein sequences from E. coli MraY, B. subtilis MraY, Homo sapiens GPT, Fission Yeast GPT, and E. coli WecA. The completely conserved amino acids are marked with an asterisk (*), and partially conserved amino acids are marked with a colon (:).

Sarah E Furlong, et al. Protein Sci. 2012 September;21(9):1366-1375.

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