Advanced

Reconstitution of water channel function and 2D-crystallization of human aquaporin 8.

Agemark, Maria LU ; Kowal, Julia; Kukulski, Wanda; Nordén, Kristina LU ; Gustavsson, Niklas LU ; Johanson, Urban LU ; Engel, Andreas and Kjellbom, Per LU (2012) In Biochimica et Biophysica Acta 1818(3). p.839-850
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 one human aquaporin (HsAQP5) has 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 HsAQP5). Thus, it is... (More)
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 one human aquaporin (HsAQP5) has 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 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. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Biochimica et Biophysica Acta
volume
1818
issue
3
pages
839 - 850
publisher
Elsevier
external identifiers
  • wos:000301155600054
  • pmid:22192778
  • scopus:84855886719
ISSN
0006-3002
DOI
10.1016/j.bbamem.2011.12.006
language
English
LU publication?
yes
id
7c90a8ad-e86e-4784-ae1b-764fb4b2e650 (old id 2273629)
date added to LUP
2012-01-12 16:59:20
date last changed
2017-04-20 13:33:49
@article{7c90a8ad-e86e-4784-ae1b-764fb4b2e650,
  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 one human aquaporin (HsAQP5) has 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 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.},
  author       = {Agemark, Maria and Kowal, Julia and Kukulski, Wanda and Nordén, Kristina and Gustavsson, Niklas and Johanson, Urban and Engel, Andreas and Kjellbom, Per},
  issn         = {0006-3002},
  language     = {eng},
  number       = {3},
  pages        = {839--850},
  publisher    = {Elsevier},
  series       = {Biochimica et Biophysica Acta},
  title        = {Reconstitution of water channel function and 2D-crystallization of human aquaporin 8.},
  url          = {http://dx.doi.org/10.1016/j.bbamem.2011.12.006},
  volume       = {1818},
  year         = {2012},
}