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In this study the x-ray structure of bovine aquaporin 0 (AQP0) was determined …

Biology Articles » Biochemistry » The channel architecture of aquaporin 0 at a 2.2-Å resolution » Materials and Methods

Materials and Methods
- The channel architecture of aquaporin 0 at a 2.2-Å resolution

AQP0 Purification, Characterization, and Crystallization. Lenses were removed from fresh bovine eyes (Harris Ranch Beef, Selma, CA), rinsed in lens buffer (0.5 M NaCl/20 mM Tris, pH 8.0/1 mM EDTA/0.5 mM PMSF), and decapsulated. The lenses were homogenized, stirred overnight, and washed five times by centrifugation at 125,000 × g for 1 h. Even though stripping the membranes with urea and alkaline buffer (14) removes most membrane protein contaminants (15); this treatment induced AQPO oligomerization upon purification (W.E.C.H., L.J.W.M., and D.A., unpublished data) and, thus, was not performed. AQP0 was purified according to ref. 16 with the following changes: 300 mM octylglucopyranoside (OG) was used for solubilization, followed by cation exchange at pH 9.0 (20 mM bicine/40 mM OG/1M NaCl elution) and size-exclusion (TSK) by using 10 mM n-nonyl-β-d-glucoside (NG) at pH 7.0 (20 mM Hepes/0.4 M NaCl). Before crystallization, the second eluted TSK peak was concentrated to 5–10 mg/ml (stirred-cell concentrator; Amicon; YM-30 membrane) and dialyzed for 3 days against 10 mM bicine, pH 9.0/10 mM NG/50 mM NaCl in a 100-KDa cut-off bag. The mass of purified bAQP0 was measured to within 1 Da of the mass calculated from the amino acid sequence by using matrix-assisted laser desorption ionization–time-of-flight MS and a sinapinic acid matrix. The radius of hydration and molar mass of bAQP0/OG protein–detergent complex were measured by combination of UV absorbance, viscosity, refractive index, and light scattering, and they were found to be 4.83 nm and 187,857 Da, respectively (Tetra-Detector analysis coupled with a TSK3000SW size-exclusion column; Viscotek, Houston). Therefore, the protein is tetrameric in solution and contains 0.66 g of OG per g of protein, or 256 mol of OG per mol of AQP0 tetramer, identical to that found by using analytical centrifugation and the anthrone method (17).

Crystallization Methods. Sitting drops were set up in a vapor-diffusion chamber by using equal volumes of protein solution and well solution (usually 2 μl of each solution) at room temperature. The well solution was 30% polyethylene glycol 1K/20 mM glycine, pH 10.0/50 mM NaCl. Although many conditions gave crystals, only one condition gave high-resolution diffracting crystals, and these crystals first appeared over the course of several months and “matured” over 1 year of incubation before the crystals diffracted to 2.20 Å. These crystals were regular tetragonal prisms of 100–200 μm in length, and they were uniaxially birefringent under polarized light.

Data Collection. Crystals were looped out of their drops and frozen by rapid immersion into liquid nitrogen. Data were collected at Beamline 8.3.1 at the Advanced Light Source (ALS) at the Lawrence Berkeley National Laboratory (Berkeley, CA). The crystals were mounted onto the synchrotron goniometer and kept at approximately –100 K in a nitrogen cryostream. Four data sets were taken from different nonoverlapping areas of one crystal. The four data sets were processed by using by denzo (18) and mosflm (19). The data used for refinement were the output of denzo/scalepack. The initial phase and solution was determined by molecular replacement using the 2.2-Å structure of bAQP1 as the search model. All subsequent refinement was carried out by using cns (20) and moloc (21).

Supporting Information. See Supporting Methods and Figs. 6–10, which are published as supporting information on the PNAS web site, for more details.


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