Electron microscopy and diffraction data are used to investigate the crystal structure of the pore forming matrix porin (ompF gene product) from the outer membrane of E. coli BE for correlations of structure and function. Existing three dimensional porin structures of negatively-stained polymorphs at 20A resolution, which clearly depict the pore channel traversing the membrane, will be extended to the maximum resolution possible with various sugar and hydrated preparations. (At present 13.7A resolution is seen in electron diffraction patterns; 8A is identified from neutron diffraction studies). This information will be sought with image data from a cryomicroscope (because of the unsuitability of high resolution electron diffraction data from slightly bent protein crystals for structure analysis via phase extension). This will allow more accurate definition of the pore channel shape and of protein domains. A comparison of image densities in variously embedded (e.g. negative stain, sugars, vitreous ice) samples will also help to identify the location of functional moieties. Quickly frozen hydrated specimens reconstituted in function altering media containing acidic phospholipid head groups and/or at differing pH's will also be studied. In relation to this, using large sugars (tri-, tetra- or penta-saccharides), constricted regions of the pore involved with gating will be identified. The spontaneous transition of a large hexagonal polymorphic form to a smaller hexagonal form via lipid loss will be evaluated in terms of possible structural changes in the protein. The phase transition between the small hexagonal and orthorhombic forms will also be investigated by a comparison of pore and protein profiles of the averaged images and also via the use of continuous diffuse low angle electron scattering in the diffraction patterns (which is normally excluded during the process of image averaging) to help model the intermediate phase. These studies strive to give a structural basis for the activity and aggregation of membrane pores, using the E. coli matrix porin as a paradigm for biological molecular sieves which restrict transport of hydrophilic species greater than MW650. Understanding this pore behavior may also give some insights into the molecular mechanism for smaller ion-selective membrane pores involved in nerve conduction.
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