The long-term objective of this application is to obtain the 3D structure of functional integral membrane proteins in their native environment and without the need for protein purification. We will focus on channels and transporters, membrane proteins that mediate a myriad of crucial cell and tissue functions. To accomplish this objective, we propose to apply freeze-fracture and shadowing, a method that replicates frozen specimens with a layer of metal ions. The immediate objective of the application is to optimize the freeze-fracture and shadowing method to produce replicas representing faithful copies of the outer shell of membrane proteins. The replicas will be studied in the electron microscope and the structure of the membrane proteins reconstructed using single particle computer image processing method. The freeze-fracture and shadowing method will be optimized by imaging purified aquaporin-0 (AQP0) and the Na+/glucose transporter (SGLT) reconstituted in liposomes. The selection of these proteins is based on our familiarity with their purification and reconstitution procedures as well as our ability to modify their structure using biochemical and molecular biology methods. Crucial to the studies proposed in this application is that two proteins of the AQP0 family, the glycerol conducting channel and aquaporin-1 (AQP1), has been solved at atomic resolution using x-ray and electron diffraction methods. These models will guide the optimization freeze-fracture and shadowing method and guarantee accurate 3D-representations of the structure of the channel. The optimized freeze-fracture and shadowing method will be used to determine the 3D structure of AQP0 and SGLT that have been modified using partial proteolysis of the C-terminus, generation of truncated and fusion proteins as well as cysteine mutagenesis and the chemistry of cysteine residues. After optimization of the freeze-fracture and shadowing method in liposomes, AQP0 and SGLT will be expressed in Xenopus laevis oocytes as a first step in determining the structure of membrane proteins directly from their cRNA, without need of protein purification.
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| Zampighi, G A; Zampighi, L; Fain, N et al. (2005) Conical tomography II: A method for the study of cellular organelles in thin sections. J Struct Biol 151:263-74 |
| Zampighi, L M; Kavanau, C L; Zampighi, G A (2004) The Kohonen self-organizing map: a tool for the clustering and alignment of single particles imaged using random conical tilt. J Struct Biol 146:368-80 |
| Zampighi, Guido A; Kreman, Michael; Lanzavecchia, Salvatore et al. (2003) Structure of functional single AQP0 channels in phospholipid membranes. J Mol Biol 325:201-10 |
