Influence of lipids on membrane protein structure and function

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DESCRIPTION (provided by applicant): 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.