The molecular structure of PhoE porin will be determined using electron crystallographic techniques. We will exploit our success in the reconstitution of membrane protein with lipid forming highly coherent 2- dimensional (2-D) crystals. We will also use specimens in an unfixed unstained condition, and new methods for obtaining flatter specimens on the electron microscope grid in the collection of a 3-dimensional (3-D) data set for high resolution structural determination of PhoE porin. Initially a 3-D structure at a resolution of about 7 Angstroms will be determined. The structure at this resolution will provide information about the pore design in the narrower segment of the channel that is not visible from our 3-D structure of the negatively strained PhoE sample. The structure at 7 Angstroms resolution is also useful for our subsequent structure determination at 3-4 Angstroms resolution. The molecular structure at this higher resolution should provide information about the detailed folding of the peptide forming the channel, and the molecular design of the channel in terms of its surface charge distribution, both of which may play an important role in regulating diffusion of ionic solutes through the channels. Information about the distribution of surface charge would also provide some understanding about the preferential permeability of negatively charged solutes and about the specificity of transport of phosphate containing-compounds. We will also determine the 3-D structure of negatively stained LamB porin at about 20 Angstroms to determine whether there is a common structural theme within the porin family. In addition, we will determine whether there is a common structural theme within the porin family. In addition, we will determine the stoichiometry of maltodextrin binding using gold-conjugated maltodextrins as a specific label. The overall goal of our structural studies is to give a rigorous conceptual framework for a rational understanding of the molecular mechanism of porins in terms of possible opening and closing of the channel and the specificity for transport of solutes across the outer membrane. The understanding of transport through the porin channel may provide an insight to the transport mechanism of other channel forming proteins.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Lawrence Berkeley National Laboratory
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