GM1 clustering inhibits cholera toxin binding in supported phospholipid membranes.

The present studies explore multivalent ligand-receptor interactions between pentameric cholera toxin B subunits (CTB) and the corresponding membrane ligand, ganglioside GM1. CTB binding was monitored on supported phospholipid bilayers coated on the walls and floors of microfluidic channels. Measurements were made by total internal reflection fluorescence microscopy (TIRFM). Apparent dissociation constants were extracted by fitting the binding data to both the Hill-Waud and Langmuir adsorption isotherm equations. Studies of the effect of ligand density on multivalent CTB-GM1 interactions revealed that binding weakened with increasing GM1 density from 0.02 mol % to 10.0 mol %. Such a result could be explained by the clustering of GM1 on the supported phospholipid membranes, which in turn inhibited the binding of CTB. Atomic force microscopy (AFM) experiments directly verified GM1 clustering within the supported POPC bilayers.