Characterizing Changes In The Structure And Orientation Of Supported Model Membranes Upon Binding Of Cholera Toxin B

Abstract

Cholera is caused by a protein toxin secreted by the bacterium Vibrio cholerae. This toxin is a member of the AB5 superfamily of toxins composed of an active (A) and a binding (B) pentameric unit. The five binding subunits of the pentamer bind specifically to the GM1, which is present in the outer leaflet of the host's plasma membrane; however, the mechanism for the translocation of the toxin is not well understood. Model membranes consisting of DMPC+cholesterol+GM1 were supported on gold electrode surfaces. The changes in the structure and orientation of the model membrane upon binding of the cholera toxin B unit were explored using differential capacitance, chronocoulometry and polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS). Changes in the structure and orientation of the toxin protein with respect to a changing electric field were similarly investigated. The IR data suggests that the binding of the toxin to the membrane causes a decrease in the number of gauche conformers, which is caused by the constriction of the acyl chains due to interactions between the toxin and the GM1 glycolipids. The bound toxin induces some minor defects in the membrane; however, these defects are not significant enough to cause measurable changes in the average orientation of the membrane lipids. The major change in the bilayer upon binding of the cholera B unit was a remarkable decrease in the relative hydration of the membrane. This decrease in hydration is mostly like due to the separation of the bilayer from the aqueous electrolyte by the presence of the bound protein layer on the membrane surface. This work is part of ongoing study to understand the mechanism for translocation of the cholera toxin across the plasma membrane.