Protein-mediated water transport is a fundamental physiological process in all organisms. It is carried out by integral membrane proteins, aquaporins, that function as water channels. Aquaporins serve as passive, diffusion-limited channels to dissipate osmotic gradients that form across cell membranes. Currently, six different human aquaporins have been discovered, each with a different distribution in bodily organs, tissues and cells. This heterogeneous distribution predicts significant roles for human aquaporins in both normal physiology and disease. Mutations in one aquaporin, aquaporin-2, are responsible for nephrogenic diabetes insipidus. Others are implicated in the maintenance of water homeostasis in erythrocytes, kidney, lung, brain and salivary gland. The aquaporins are likely targets for the future development of therapeutic agents directed to prevention or control of edema and fluid balance. The molecular basis of protein-mediated water transport will be elucidated by crystal structure determination of human aquaporins. Purification of aquaporin- 1 (AQP- I), the archetypal aquaporin, from erythrocytes yields quantities of protein suitable for crystallization. Crystals that diffract to 3.5A have been obtained, and a complete 4A dataset has been collected. Mercury/AQP-1 co-crystals have been obtained, and will be used in the determination of the structure of AQP-1 by multiple isomorphous replacement. Models derived from electron crystallography of two-dimensional crystals will also be used to assist in phase determination. The structure at 4A resolution will define the architecture of the aqueous pore and describe the protein fold. Next, crystals of AQP-I will be improved for the determination of a complete structure at higher resolution. Lastly, recombinant aquaporins other than AQP-1 will be produced for crystallization experiments. The ultimate goal is the determination of an ensemble of human aquaporin structures for use in structure- based drug design.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM056251-04
Application #
6181253
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Lewis, Catherine D
Project Start
1997-08-01
Project End
2002-07-31
Budget Start
2000-08-01
Budget End
2001-07-31
Support Year
4
Fiscal Year
2000
Total Cost
$196,533
Indirect Cost
Name
University of Virginia
Department
Physiology
Type
Schools of Medicine
DUNS #
001910777
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Mohanty, Arun K; Wiener, Michael C (2004) Membrane protein expression and production: effects of polyhistidine tag length and position. Protein Expr Purif 33:311-25
Mohanty, Arun K; Simmons, Chad R; Wiener, Michael C (2003) Inhibition of tobacco etch virus protease activity by detergents. Protein Expr Purif 27:109-14
Purdy, Michael D; Ge, Pinghua; Chen, Jiyan et al. (2002) Thiol-reactive lanthanide chelates for phasing protein X-ray diffraction data. Acta Crystallogr D Biol Crystallogr 58:1111-7
Wiener, M C (2000) Bacterial export takes its Tol. Structure 8:R171-5
Wiener, M C; Verkman, A S; Stroud, R M et al. (2000) Mesoscopic surfactant organization and membrane protein crystallization. Protein Sci 9:1407-9
Purdy, M D; Wiener, M C (2000) Expression, purification, and initial structural characterization of YadQ, a bacterial homolog of mammalian ClC chloride channel proteins. FEBS Lett 466:26-8
Kleinschmidt, J H; Wiener, M C; Tamm, L K (1999) Outer membrane protein A of E. coli folds into detergent micelles, but not in the presence of monomeric detergent. Protein Sci 8:2065-71