Folding andBiophysical Characterization of Neisserial Outer Membrane Opacity-associated (Opa) Proteins in Lipid Vesicles

Dewald, Alison Hodge, Department of Chemistry, University of Virginia
Columbus, Linda, Department of Chemistry, University of Virginia
Cafiso, David, Department of Chemistry, University of Virginia
Bryant, Robert, Department of Chemistry, University of Virginia
Lehman, Kevin, Department of Chemistry, University of Virginia
Criss, Alison, Department of Microbiology, University of Virginia

This dissertation presents the folding and characterization of Opacity-Associated (Opa) proteins in lipid membranes. Opa are -barrel outer membrane proteins (OMPs) of the bacterial pathogens Neisseria gonorrhoea and N. meningiditis. When Opa interact with select human host cell receptors, they trigger the engulfment of the bacterium by human cells. There are hundreds of Opa variants, with differing receptor specificity. The specificity is attributed to differences in the proteins' highly variable extracellular loops. The spontaneous folding of two variants, Opa 60 and Opa 50, into liposomes was investigated by systematically varying bulk and membrane properties. Centrifugal fractionation coupled with SDS-PAGE enabled the discrimination of aggregate, unfolded membrane-associated, and folded membrane-inserted protein states and the influence of pH, ionic strength, membrane surface potential, lipid saturation, and urea on each. Protein aggregation was reduced with increasing lipid chain length, basic pH, low salt, the incorporation of negatively charged lipids, or the addition of urea to the folding reaction. Insertion from the membrane-associated form was improved in shorter chain lipids, with basic pH, and low ionic strength; and was hindered by unsaturated or ether-linked lipids. Membrane surface and dipole potentials were identified as driving forces for OMP insertion and folding into lipid bilayers. The Opa proteoliposomes were used to assess the binding of Opa 60 and Opa 50 with their cognate receptors. A lipid mixture of DMPC, DMPG, and cholesterol was identified for increased liposome stability and a sonication-based method was developed to transfer folded Opa protein into the stable proteoliposomes. Trypsin digestion was used with SDS-PAGE to identify the orientation of Opa 60 in liposomes as half outward. Opa 60 and x Opa 50 bound their cognate receptors, and a pull-down assay was developed towards elucidating the thermodynamics of Opa-receptor binding. The Opa proteoliposome system allows the study of Opa protein function in isolation from other cellular components in a quantitative, controlled way and may therefore be used to elucidate the thermodynamics and molecular determinants of Opa - receptor binding. Knowledge of these determinants may be of use for developing treatment for Neisseria related disease, or for the design of Opa-decorated proteoliposomes for targeted, intracellular drug delivery.

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PHD (Doctor of Philosophy)
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