Reconstitution of water channel function and 2D-crystallization of human aquaporin 8.
(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:
https://lup.lub.lu.se/record/2273629
- author
- Agemark, Maria LU ; Kowal, Julia ; Kukulski, Wanda ; Nordén, Kristina LU ; Gustavsson, Niklas LU ; Johanson, Urban LU ; Engel, Andreas and Kjellbom, Per LU
- organization
- publishing date
- 2012
- 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
- pmid:22192778
- 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
- 2016-04-01 14:15:19
- date last changed
- 2022-02-12 01:34:07
@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}}, doi = {{10.1016/j.bbamem.2011.12.006}}, volume = {{1818}}, year = {{2012}}, }