An understanding of protein translocation and import across receptors and membranes is fundamental in biology and medicine. Receptor-mediated import of the toxin-like colicins is a paradigm for protein import to mitochondria and the import of viral proteins. Colicins E1 and E3 have different modes of bactericidal action, but parasitize the same (vitamin B12, BtuB) integral outer membrane receptor, and are used to study the mechanism of import in the E. coil inner and outer membrane, respectively. (I) OM import: (1) A 2.75 A structure of the complex of the expressed 135 residue (R135) 100 A long coiled-coil receptor binding domain of colicin E3, and the BtuB receptor in detergent, is the first high resolution structure of an intact integral protein receptor and its protein ligand. This structure forms the basis for studies on mechanisms of protein translocation through the outer membrane. (2) SPR and CD show that R135 binds tightly, and its receptor-distal ends unwind upon binding to BtuB. A smaller R60 peptide rewinds upon binding. The basis for these unusual properties of the binding site will be probed by mutagenesis of both R135 and BtuB, particularly the loop regions of BtuB. An R135 Arg399Ala mutant has defined a residue involved in electrostatic binding. Disulfide cross-linking of the distal ends of R135 will test the coupling between distal unwinding and binding affinity. (3) The 2.75 A structure and the absence of large ion channels in BtuB imply that E3 does not pass through it. Occlusion of the large OmpF and TolC ion channels by E3 and E1, respectively, indicate a role for these Omp in the colicin translocon. (4) The bending and mechanical properties of the R135 coiled-coil, which might be involved in delivery of colicin domains to OmpF, will be tested by atomic force microscopy. (5) The role of two OM receptors in colicin translocation will be tested with OmpF and TolC, (i) using planar bilayers to measure changes in their channel conductance and receptor function, and the ability of individual colicin domains and TolA, TolB proteins to occlude the channels; and (ii) in co-crystallization experiments. (II) IM import: We have proposed that two prerequisites for import of soluble membrane-active proteins are segmental mobility on the membrane surface and helix extension. (1) This hypothesis will be further tested using (a) the colicin channel protein, P178, membranes of defined surface potential, and assays by FRET measurements of distance changes, and (b) solid-state NMR. (2) The pathway of membrane insertion of the E1 channel domain, relative to the time course of other unfolding events, will be examined by time-resolved FRET using a large set of single Trp mutants. ? ?
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