Functional reconstitution and characterization of AqpZ, the E-coli water channel protein

Understanding the selectivity of aquaporin water channels will require stru ctural and functional studies of wild-type and modified proteins; however, expression systems have not previously yielded aquaporins in the necessary milligram quantities. Here we report expression of a histidine-tagged form of Escherichia coli aquaporin-Z (AqpZ) in its homologous expression system. 10-His-AqpZ is solubilized and purified to near homogeneity in a single st ep with a final yield of similar to 2.5 mg/l of culture. The histidine tag is removed by trypsin, yielding the native protein with the addition of thr ee N-terminal residues, as confirmed by microsequencing. Sucrose gradient s edimentation analysis showed that the native, solubilized AqpZ protein is a trypsin-resistant tetramer. Unlike other known aquaporins, AqpZ tetramers are not readily dissociated by 1% SDS at neutral pH. Hydrophilic reducing a gents have a limited effect on the stability of the tetramer in 1% SDS, whe reas incubations for more than 24 hours, pH values below 5.6, or exposure t o the hydrophobic reducing agent ethanedithiol cause dissociation into mono mers. Cys20, but not Cys9, is necessary for the stability of the AqpZ tetra mer in SDS. Upon reconstitution into proteoliposomes, AqpZ displays very hi gh osmotic water permeability (p(f) greater than or equal to 10 x 10(-14) c m(3) s(-1) subunit(-1)) and low Arrhenius activation energy (E-a = 3.7 kcal /mol), similar to mammalian aquaporin-1 (AQP1). No permeation by glycerol, urea or sorbitol was detected. Expression of native and modified AqpZ in mi lligram quantities has permitted biophysical characterization of this remar kably stable aquaporin tetramer, which is being utilized for high-resolutio n structural studies. (C) 1999 Academic Press.