Harries et al. 10.1073/pnas.0405274101.
Fig. 6. Alignment of all of the AQPs that have had their high-resolution structures determined and AQP0 human. From the top in descending order: GlpF-E.coli, AQP0-Bovine, AQP0-Human, AQP1-Bovine, AQPZ-E.coli. This alignment was run by the CLUSTALW program. CLUSTALW Service at the European Bioinformatics Institute is available at http://www.ebi.ac.uk/clustalw.
Fig. 7. Multipanel AQP0 Purification Results. From left to right, the first image is a photograph of a 4–20% Tris·glycine/PAGE gel, summarizing the purification process of bovine AQP0. Lane 1 shows the molecular weight standards at 6, 14.4, 21.5, 31, 36.5, 55.4, 66.3, 97.4, 116.3, and 200 kDa (from bottom to top). Lane 2 shows the postsolubilization before spin sample. Lane 4 shows the postsolubilization after spin sample. Comparisons of lanes 2 and 4 illustrate the efficacy of detergent solubilization. Lane 3 is the protein profile after the HiTrap Sp column. Lane 5 is the protein profile of pooled third peaks from the TSK size-exclusion column and shows a high level of purification (» 95% pure bovine AQP0). Lane 6 is the protein profile of the bovine AQP0 preparation after purification, concentration, and dialysis. This material went to crystal trials. The second image shows the elution profile of bovine AQP0 from the HiTrap Sp column. The peak after the high-salt bump contains AQP0. The third image shows the elution profile from the TSK size-exclusion column. The third major peak contains the tetrameric AQP0. The fourth image shows the elution profile of our purified, concentrated, and dialyzed AQP0 sample that is ready for crystal trails. The absence of significant other peaks indicates that the sample is primarily tetrameric with little oligomerization and is, thus, stable throughout our experimental protocol.
Fig. 8. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS plot of the after-purification/precrystallization bovine AQP0 preparation. The expected molecular mass derived from the amino acid sequence in 28,222.9, and the observed molecular mass is 28,228.26. The MALDI-TOF matrix had the following components: 10 mg of sinapinic acid, 10 ml of 3% trifluoracetic acid, 500 m l of acetronitrile, and 400 ml of H2O. Purified and concentrated AQP0 protein was added to the matrix mixture in a 1:9 protein/matrix mixture.
Fig. 9. Bovine AQP0 Crystallography. Upper Left shows decapsulated bovine lenses before AQP0 purification. Lower Left shows a bovine AQP0 crystal in its growth drop, showing birefringence under polarizing optics. Right shows a false-color image of the bovine AQP0 diffraction pattern. Spots at the outer periphery correspond to a resolution of 2.2 Å.
Fig. 10. LIGPLOT diagram of the interactions of bovine AQP0 extracellular domains with the double-layered membrane configuration of the electron-diffraction structure. No hydrogen bonds are detected between opposed AQP0 extracellular domains. Only hydrophobic interactions are suggested by the LIGPLOT program (1).
Polymeric and hydrodynamic analysis of purified bovine AQP0 using MS and Tetra-Detector analytical methods.