CONFORMATIONAL-CHANGES IN CHOLERA-TOXIN-B SUBUNIT GANGLIOSIDE-GM1 COMPLEXES ARE ELICITED BY ENVIRONMENTAL PH AND EVOKE CHANGES IN MEMBRANE-STRUCTURE

Fluorescence resonance energy transfer (FRET) was used to monitor pH-d ependent structural changes in the cholera toxin B subunit (CTB) and t he membranes with which CTB associates. The distance separating the si ngle tryptophan (Trp88) of each CTB monomer and a pyrene probe linked to the membrane-imbedded tail of ganglioside GM1 is not influenced by pH in a range from 3.5 to 7.5, consistent with the position of Trp88 i n the GM1 binding site of CTB. In contrast, the distance between the p yrene probe on GM1. and coumarin, stilbene, or fluorescein probes cova lently linked to specific sites on CTB appears to increase significant ly as the pH is lowered to 5.0 or less. This conformational change is not accompanied by detectable changes in the distance between Trp88 an d these extrinsic probe positions in the presence of nonfluorescent GM 1. However, when the distance from Trp88 to the extrinsic probes is mo nitored as a function of pH in the absence of GM1, a conformational ch ange is seen which indicates that receptor binding influences the char acter of pH-dependent conformational changes that occur within CTB. In terestingly, the observed change in CTB conformation is accompanied by a change in the relative position of GM1 within the membrane as judge d by FRET from the pyrene probe on GM1 to a 7-nitrobenz-2-oxa-1,3-diaz ol-4-yl (NBD) probe linked to the polar head group of phosphatidyletha nolamine and positioned at the membrane surface. Taken together, the d ata imply that low endosomal pH is capable of inducing structural chan ges in CTB, which, in turn, exert effects on the structure of the memb rane to which CTB is bound. These phenomena may have a role in (1) pro cessing of cholera toxin within the endosomal compartments of some tar get cell types, (2) determining the lag time between cholera toxin bin ding and the target cell response to cholera intoxication, or (3) the efficiency of CTB and cholera toxin as mucosal adjuvants.