Multivalent interactions are central to biological processes such as immune responses and viral entry, and their study is crucial for targeted drug development and biosensor design. However, most experimental investigations are conducted at receptor densities higher than physiological levels, while studies at lower densities often cannot be captured by existing binding models. Here, we constructed supported lipid bilayers containing synthetic lipids doped with GM1 gangliosides at controlled densities, ranging from physiologically relevant low densities to the higher densities commonly used in prior work. We introduced fluorescently labeled cholera toxin B subunit (CTB) to these membranes and measured equilibrium binding by quantifying fluorescence intensity. This enabled systematic characterization of CTB-GM1 multivalent binding across a broad density spectrum. Our results reveal that at physiological low receptor densities, CTB exhibits equilibrium binding behavior opposite to that observed at high densities. We attribute this counterintuitive trend to steric blocking of receptors by CTB, together with crowding effects that constrain multivalent binding. To account for these effects, we developed a new model that incorporates steric crowding into existing receptor-blocking frameworks. Comparison with experimental results demonstrates that the model successfully reproduces conventional binding at high densities while also predicting the unusual low-density behavior. We believe that this unique binding phenomenon and model provide new perspectives and valuable guidance for extending targeted drug applications and biosensor design to lower receptor density regimes.
Hsu et al. (Sun,) studied this question.