title: Reconstitution of water channel function and 2D-crystallization of human aquaporin 8.
abstract: Among the thirteen human aquaporins (AQP0-12), the primary structure of AQP8 is unique. By sequence alignment it is evident that mammalian AQP8s form a separate subfamily distinct from the other mammalian aquaporins. The constriction region of the pore determining the solute specificity deviates in AQP8 making it permeable to both ammonia and H(2)O(2) in addition to water. To better understand the selectivity and gating mechanism of aquaporins, high-resolution structures are necessary. So far, the structure of three human aquaporins (HsAQP1, HsAQP4, and HsAQP5) have been solved at atomic resolution. For mammalian aquaporins in general, high-resolution structures are only available for those belonging to the water-specific subfamily (including HsAQP1, HsAQP4 and HsAQP5). Thus, it is of interest to solve structures of other aquaporin subfamily members with different solute specificities. To achieve this the aquaporins need to be overexpressed heterologously and purified. Here we use the methylotrophic yeast Pichia pastoris as a host for the overexpression. A wide screen of different detergents and detergent-lipid combinations resulted in the solubilization of functional human AQP8 protein and in well-ordered 2D crystals. It also became evident that removal of amino acids constituting affinity tags was crucial to achieve highly ordered 2D crystals diffracting to 3Å.
authors.affiliation: Department of Biochemistry and Structural Biology, Lund University, Lund, Sweden.
authors.forename: Maria
authors.initials: M
authors.lastname: Agemark
authors.forename: Julia
authors.initials: J
authors.lastname: Kowal
authors.forename: Wanda
authors.initials: W
authors.lastname: Kukulski
authors.forename: Kristina
authors.initials: K
authors.lastname: Nordén
authors.forename: Niklas
authors.initials: N
authors.lastname: Gustavsson
authors.forename: Urban
authors.initials: U
authors.lastname: Johanson
authors.forename: Andreas
authors.initials: A
authors.lastname: Engel
authors.forename: Per
authors.initials: P
authors.lastname: Kjellbom
mesh_terms: D020346:Aquaporins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D018360:Crystallography, X-Ray; D003902:Detergents / Q000737:chemistry; D015870:Gene Expression; D006801:Humans; D008055:Lipids / Q000737:chemistry; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D013329:Structure-Activity Relationship
chemical_list: D020346:Aquaporins; D003902:Detergents; D008055:Lipids; D011994:Recombinant Proteins; C108330:aquaporin 8