College Park, Maryland      June 6 - 10 , 2004

MP51: Cholera Toxin Assault on Lipid Monolayers Containing Ganglioside GM1 : a Neutron and X-Ray Scattering Study at the Air-Liquid Interface

C.E. Miller (Biophysics Graduate Group, University of California, Davis; Department of Chemical Engineering and Material Science, University of California, Davis), J. Majewski (Los Alamos Neutron Science Center, Los Alamos National Laboratory), K. Kjær, M. Weygand (Physics Department, Risø National Laboratory, Denmark), R. Faller (Department of Chemical Engineering and Material Science, University of California, Davis), T.L. Kuhl (Department of Chemical Engineering and Material Science, University of California, Davis; Biophysics Graduate Group, University of California, Davis)

Many bacterial toxins bind to and gain entrance to target cells through specific interactions with membrane components. Using neutron/x-ray reflectivity and x-ray grazing incidence diffraction (GID), we have characterized the structure of mixed DPPE:GM1 lipid monolayers before and during the binding of cholera toxin (CTAB5) or its B subunit (CTB5). Structural parameters such as the density and thickness of the lipid layer, extension of the GM1 oligosaccharide headgroup, and orientation and position of the protein upon binding are reported. The density of the lipid layer was found to decreases slightly upon protein binding. However, the alpha subunit of the whole toxin is clearly located below the B pentameric ring, away from the monolayer, and does not penetrate into the lipid layer prior to enzymatic cleavage. Using Monte Carlo simulations, the observed monolayer expansion was found to be consistent with geometrical constraints imposed on DPPE by multivalent binding of GM1 by the toxin. Our findings suggest that the mechanism of membrane translocation by the protein may be aided by alterations in lipid packing. Both CTAB5 and CTB5 were measured to have ~50 % coverage when bound to the lipid monolayer. X-ray GID experiments show that both the lipid monolayer and the cholera toxin layer are crystalline. The effects of x-ray beam damage have been assessed and the monolayer/toxin structure does not change with time after protein binding has saturated.

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