Water transport regulation: Biochemical and structural analyses of eukaryotic aquaporins
Doktorsavhandling, 2008

Water is essential for all living beings. Aquaporins are water channel proteins embedded in the membranes of cells. These macromolecules are found in all kingdoms of life and they control water homeostasis by diverse regulation mechanisms. Increased structural and functional knowledge of aquaporins will aid in understanding how the flux of water through these pores is controlled and why certain physical disorders in humans connected to the aquaporins arise. Large quantities of protein are required for structural analysis. Since many eukaryotic membrane proteins are not abundant in nature, reliable protein overproduction systems are a prerequisite for further advancement of the field. In this thesis the Pichia pastoris overproduction system was evaluated for heterologous production of all the thirteen human aquaporins. Our findings show that aquaporins, even though similar in size and fold, give very diverse protein yields. Factors influencing the level of protein production are discussed, and one construct of human aquaporin 1 resulted in an exceptionally high yield. Observations regarding the influence of the aquaporin construct on crystal packing are also discussed. Regulatory mechanisms of the spinach aquaporin SoPIP2;1 were also investigated. To mimic phosphorylation, residues implicated in the opening and closing of this gated aquaporin were mutated and their X-ray structures elucidated. Our findings show that the mutation of serine 115 to a glutamate led to a half turn extension of transmembrane helix one into the cytoplasm. Furthermore, serine 188 was identified as a putative phosphorylation site as its mutation to a glutamate increased the water flux through the aquaporin. These findings provide further insight into the molecular gating mechanism previously suggested for this plant aquaporin.

X-ray crystallography

regulation

aquaporins

function

structure

membrane proteins

KB Kemigården 4
Opponent: Christophe Maurel

Författare

Anna Maria Nyblom

Chalmers, Kemi- och bioteknik, Molekylär mikroskopi

Aquaporin gating

Current Opinion in Structural Biology,; Vol. 16(2006)p. 447-456

Artikel i vetenskaplig tidskrift

Ämneskategorier

Biokemi och molekylärbiologi

Strukturbiologi

ISBN

978-91-7385-090-2

Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 2771

KB Kemigården 4

Opponent: Christophe Maurel