NIH-R01-GM066988: Export & Import of Lethal Agents Mediated by TolC (2003 – 2007)

Transport of molecules across biological membranes is a fundamental process shared by all living cells.  In Gram-negative bacteria, such as Escherichia coli, transport occurs across two membranes, of which the outer membrane represents the first permeability barrier.  Outer membrane protein-mediated transport is needed to bring nutrients inside and expel unwanted metabolites and inhibitors.  Ironically, toxic biomolecules and infectious agents, present in the hostile environment in which bacteria live, frequently exploit these outer membrane proteins to gain entry into the cell.

The focus of this research is a unique outer membrane protein, TolC, which mediates the diffusion of a diverse group of molecules including the export of α-hemolysin and the efflux of antibiotics.  Despite the resolution of TolC’s three-dimensional structure, the role of individual residues and the mechanism by which it accomplishes the transport of a diverse group of molecules remains poorly understood.  Moreover, it is unknown how TolC interacts with other proteins of the inner membrane to form the complexes that facilitate the two-way transport activity.  The present study is being undertaken to examine the role of TolC in export (antibiotic) and import (colicin E1), as well as to examine its interactions with other proteins of the transport complexes.  Identification of functionally critical TolC residues and the various protein-protein interactions that must occur to accomplish the diverse transport activities will be achieved through employing a combination of genetic, structural, and biochemical approaches; available preliminary data concerning all aspects of the project strongly reflect their feasibility.

The presence of TolC and its homologues in other Gram-negative bacteria demonstrates the broader relevance of this unique multifunctional protein.  Besides the obvious role of TolC in antibiotic resistance, its ability to mediate the secretion of a variety of virulence factors in pathogenic bacteria makes it a medically important subject of investigation.

NIH-R01-GM48167: Assembly of Outer Membrane Proteins in Escherichia coli (1992 – 2007)

Protein folding and assembly are central biological processes that must occur correctly for the proper functioning of all living cells.  Many devastating human diseases, including neurodegenerative Alzheimer and Prion diseases, are the consequence of disarrayed protein folding and assembly.  A relatively large amount of cellular activity is dedicated to ensure the correct folding and assembly of proteins.  In the event of misfolding, proteins are driven to aggregation and degradation pathways.  Degradation of misfolded proteins is crucial because they may form toxic aggregates, which can interfere with normal cellular functions.  Assembly factors that minimize aggregation (chaperones and foldases) or remove aggregates (proteases) are therefore complementary cellular activities that are regulated in response to the protein-folding status of the cell.