In membranes, proteins and lipids form an integrated system to support essential cell functions. The structure of membrane proteins embedded in native lipid bilayers can be studied by electron crystallography. Aquaporin 0 (AQPO) is the major protein constituent in lens fiber cells where it serves a dual role: creating a pore for water and forming adhesive junctions. Using electron crystallography, we have recently determined the structure of AQPO to an unprecedented resolution of 1.9A, revealing the precise structure of a shell of ordered lipids surrounding the AQPO tetramer. AQPO therefore provides the first eukaryotic model system in which it is possible to study, in the very same membrane, the structural and functional interdependence of protein and lipids. To do this, we will form single-layered two-dimensional crystals of AQPO in lipid bilayers using native or unnatural lipids and in the presence or absence of calmodulin (CaM) which binds to the C- terminal domain of AQPO. We will then correlate the structure (from electron crystallography) and function (from electrophysiology) of the AQPO water channel as it opens and closes in response to pH, divalent calcium, and other ligands. Finally, we will also determine the structural rearrangements required for pore closure when two AQPO tetramers on separate bilayers come together to form intercellular junctions. Although this proposal addresses specific questions regarding AQPO water channel function, many of the resulting insights should be generally applicable to other channels and membrane proteins. ? ? ?

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Biochemistry and Biophysics of Membranes Study Section (BBM)
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Chin, Jean
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University of Washington
Schools of Medicine
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Stokes, David L; Ubarretxena-Belandia, Iban; Gonen, Tamir et al. (2013) High-throughput methods for electron crystallography. Methods Mol Biol 955:273-96
Reichow, Steve L; Clemens, Daniel M; Freites, J Alfredo et al. (2013) Allosteric mechanism of water-channel gating by Ca2+-calmodulin. Nat Struct Mol Biol 20:1085-92
Gonen, Tamir (2013) The collection of high-resolution electron diffraction data. Methods Mol Biol 955:153-69
Wisedchaisri, Goragot; Gonen, Tamir (2013) Phasing electron diffraction data by molecular replacement: strategy for structure determination and refinement. Methods Mol Biol 955:243-72
Gold, Matthew G; Reichow, Steve L; O'Neill, Susan E et al. (2012) AKAP2 anchors PKA with aquaporin-0 to support ocular lens transparency. EMBO Mol Med 4:15-26
Wisedchaisri, Goragot; Reichow, Steve L; Gonen, Tamir (2011) Advances in structural and functional analysis of membrane proteins by electron crystallography. Structure 19:1381-93
Wisedchaisri, Goragot; Gonen, Tamir (2011) Fragment-based phase extension for three-dimensional structure determination of membrane proteins by electron crystallography. Structure 19:976-87
Jehle, Stefan; Vollmar, Breanna S; Bardiaux, Benjamin et al. (2011) N-terminal domain of alphaB-crystallin provides a conformational switch for multimerization and structural heterogeneity. Proc Natl Acad Sci U S A 108:6409-14
Wisedchaisri, Goragot; Dranow, David M; Lie, Thomas J et al. (2010) Structural underpinnings of nitrogen regulation by the prototypical nitrogen-responsive transcriptional factor NrpR. Structure 18:1512-21
Zheng, Hongjin; Taraska, Justin; Merz, Alexey J et al. (2010) The prototypical H+/galactose symporter GalP assembles into functional trimers. J Mol Biol 396:593-601

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