title: The aquaporin sidedness revisited.
abstract: Aquaporins are transmembrane water channel proteins, which play important functions in the osmoregulation and water balance of micro-organisms, plants, and animal tissues. All aquaporins studied to date are thought to be tetrameric assemblies of four subunits each containing its own aqueous pore. Moreover, the subunits contain an internal sequence repeat forming two obversely symmetric hemichannels predicted to resemble an hour-glass. This unique arrangement of two highly related protein domains oriented at 180 degrees to each other poses a significant challenge in the determination of sidedness. Aquaporin Z (AqpZ) from Escherichia coli was reconstituted into highly ordered two-dimensional crystals. They were freeze-dried and metal-shadowed to establish the relationship between surface structure and underlying protein density by electron microscopy. The shadowing of some surfaces was prevented by protruding aggregates. Thus, images collected from freeze-dried crystals that exhibited both metal-coated and uncoated regions allowed surface relief reconstructions and projection maps to be obtained from the same crystal. Cross-correlation peak searches along lattices crossing metal-coated and uncoated regions allowed an unambiguous alignment of the surface reliefs to the underlying density maps. AqpZ topographs previously determined by AFM could then be aligned with projection maps of AqpZ, and finally with human erythrocyte aquaporin-1 (AQP1). Thereby features of the AqpZ topography could be interpreted by direct comparison to the 6 A three-dimensional structure of AQP1. We conclude that the sidedness we originally proposed for aquaporin density maps was inverted.
authors.affiliation: M. E. Müller Institute for Microscopy at the Biozentrum, University of Basel, Klingelbergstr. 70, Basel, CH-4056, Switzerland.
authors.forename: S
authors.initials: S
authors.lastname: Scheuring
authors.forename: P
authors.initials: P
authors.lastname: Tittmann
authors.forename: H
authors.initials: H
authors.lastname: Stahlberg
authors.forename: P
authors.initials: P
authors.lastname: Ringler
authors.forename: M
authors.initials: M
authors.lastname: Borgnia
authors.forename: P
authors.initials: P
authors.lastname: Agre
authors.forename: H
authors.initials: H
authors.lastname: Gross
authors.forename: A
authors.initials: A
authors.lastname: Engel
mesh_terms: D051398:Aquaporin 1; D020346:Aquaporins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*; D001789:Blood Group Antigens; D020285:Cryoelectron Microscopy; D003460:Crystallization; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D005612:Freeze Drying; D006801:Humans; D008565:Membrane Proteins; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D016626:Shadowing Technique, Histology; D013499:Surface Properties; D014357:Trypsin / Q000378:metabolism
chemical_list: C496020:AQP1 protein, human; D020346:Aquaporins; D001789:Blood Group Antigens; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; C106425:aqpZ protein, E coli; D051398:Aquaporin 1; D014357:Trypsin