The crystallization of transmembrane channel-forming proteins (porins) from the outer membrane of enteric (gram negative) bacteria (e.g., E. coli and Salmonella typhimurium) create a unique opportunity to study the roles these proteins play in bacterial membrane structure and function. A high resolution (less than 2.9 angstroms) X-ray structure determination of the OMpF porin from E. coli is a major focus of the research effort. This structure would provide information on how a transmembrane protein, consisting primarily of beta-sheet structure, folds and inserts into biological membranes. It would also allow the analysis, at an atomic level, of the topography of the transmembrane pore surface and of the mechanism of ion selectivity and channel-gating. Experiments are planned to crystallize and study the OmpC porin to understand the physical aspects of the OmpF/OmpC osmoregulation system in E. coli by analyzing the effects of amino acid changes on the pore size and structure. The primary and secondary structure similarities between these and other porins permit the use of molecular replacement and averaging techniques to determine subsequent X- ray structures. The porins LamB and PhoE which display specificity towards maltose and phosphate compounds, respectively, have also been crystallized. Their X-ray structure analyses are part of an effort to study the structural basis for solute selectivity in these specific transport systems. Though the research is on bacterial passive transport systems, there will be structural implications for the function of active membrane transport in eukaryotic organisms. Furthermore, understanding the mechanism of solute selectivity and transport across the bacterial outer membrane will assist the design of antibiotics which will easily cross the outer membrane. The bacterial glycolipid lipopolysaccharide binds specifically to many of the porins. Stoichiometric complexes between lipopolysaccharide precursors and OmpF porin have been crystallized and are suitable for a high resolution X-ray analysis. A major effort is directed towards the study of the lipid binding site at atomic resolution to discern the modes of interactions between the protein and lipid. The X-ray studies will be done in conjuction with low resolution (15-16 Angstroms) neutron diffraction on single crystals and D2O/H2O density matching experiments to determine the general topography of the membrane-protein interface.
The aim i s to understand the role that the proteins and lipids play in creating a defensive barrier against adverse environments, in providing binding sites for the attachment of bacteriophages and bacterial-targeted toxins (e.g., colicins) and in eliciting immune responses during enteric bacterial infections.
|Kim, H (1998) Crystallization of OmpC osmoporin from Escherichia coli. Acta Crystallogr D Biol Crystallogr 54:1399-400|
|Garavito, R M (1994) The X-ray structures of Escherichia coli porin OmpF and ovine prostaglandin synthase: implications for membrane structure. Ren Physiol Biochem 17:219-22|
|Lal, R; Kim, H; Garavito, R M et al. (1993) Imaging of reconstituted biological channels at molecular resolution by atomic force microscopy. Am J Physiol 265:C851-6|