The aquaporin sidedness revisited

Aquaporins are transmembrane water channel proteins, which play important f unctions in the osmoregulation and water balance of microorganisms, plants, and animal tissues. All aquaporins studied to date are thought to be tetra meric assemblies of four subunits each containing its own aqueous pore. Mor eover, the subunits contain an internal sequence repeat forming two obverse ly symmetric hemichannels predicted to resemble an hour-glass. This unique arrangement of two highly related protein domains oriented at 180 degrees t o each other poses a significant challenge in the determination of sidednes s. Aquaporin Z (AqpZ) from Escherichia coli was reconstituted into highly o rdered two-dimensional crystals. They were freeze-dried and metal-shadowed to establish the relationship between surface structure and underlying prot ein density by electron microscopy. The shadowing of some surfaces was prev ented by protruding aggregates. Thus, images collected from freeze-dried cr ystals that exhibited both metal-coated and uncoated regions allowed surfac e relief reconstructions and projection maps to be obtained from the same c rystal. Cross-correlation peak searches along lattices crossing metal-coate d and uncoated regions allowed an unambiguous alignment of the surface reli efs to the underlying density maps. AqpZ topographs previously determined b y AFM could then be aligned with projection maps of AqpZ, and finally with human erythrocyte aquaporin-1 (AQP1). Thereby features of the AqpZ topograp hy could be interpreted by direct comparison to the 6 Angstrom three-dimens ional structure of AQP1. We conclude that the sidedness we originally propo sed for aquaporin density maps was inverted. (C) 2000 Academic Press.