Water and glycerol channels of the MIP family: structure, function, regulation and exploitation

• Research unit: EU RFP
• Project number: 98.0213

Key words (English)

Aquaporins; aquaglyceroporins; MIP family of proteins;electron crystallography; atomic force microscopy; sequence analysis

Alternative project number (English)

EU project number: BIO4CT980024

Research programs (English)

EU-programme: 4. Frame Research Programme - 4.1 Biotechnology

Short description (English)

See abstract

Further information (English)

Full name of research-institution/enterprise:
Universität Basel
Biozentrum, C-CINA, WRO-1058.P.24
Maurice E. Müller Institute for Structural Biology, c/o Syngenta AG

Partners and International Organizations (English)

Coordinator: Universität Göteborg (S)

Abstract (English)

The Basel team gathered a large variety of structural information from different channel proteins that were produced within the consortium. Purification protocols were optimized, and native and heterologously expressed proteins were tested for their propensity to form 2D crystals, and were analyzed by atomic force microscopy and electron crystallography.
In collaboration with Dr. Fujiyoshi, Kyoto University, the atomic structure of AQP1 was solved. 2D crystals produced in Basel were used for electron crystallography. Sequence analyses (Basel team) and fitting of helical segments to the 4.5 A structure (Goettingen team) provided a solid basis for the model building in Japan.
The Basel and Lund teams discovered a new isoform of spinach water channels, PM28C. Both PM28A, the channel previously identified, and PM28C were characterized by mass-spectrospy, atomic force microscopy, and negative stain electron microscopy, and found to be tetramers. These preparations showed a propensity to form 2D crystals.
AQP2 was produced in Nijmegen in SF9 cells in mg-quantities. This protein exhibited native water transport capacity in reconstituted vesicles. Both the native and the his-tagged recombinant proteins showed identical water transport. An efficient AQP2 purification protocol and 2D crystallization procedure were developed. These crystals diffract to 7 Å.
Excellent GlpF crystals were grown in Basel and the 3.7 A projection map as well as a 3D map at 6.9 A were established. The prominent structural homology with the AQP1 molecule fostered homology modelling based on an extended sequence analysis. The essential features of the glycerol channels could be established, although the stereospecificity can only be explained by higher resolution data now available from X-ray crystallography.

References in databases (English)

Swiss Database: Euro-DB of the
State Secretariat for Education and Research
Hallwylstrasse 4
CH-3003 Berne, Switzerland
Tel. [phone]
Swiss Project-Number: 98.0213