NECEEM-Enabled Mechanistic Analysis of Cooperative Binding: Application to C-Reactive Protein–SOMAmer Complexes

ABSTRACT: Understanding cooperative binding is essential for characterizing interactions between multimeric proteins and their ligands, as their biological function may depend on binding stoichiometry or allosteric regulation, which in turn requires determining the individual dissociation constants for each binding step (Kd1, Kd2, etc.). However, most experimental methods rely on ensemble-averaged signals that cannot resolve coexisting complexes, forcing stepwise thermodynamic parameters to be inferred through model-dependent fitting procedures that cannot be independently validated. To date, detailed determinations of Kd1 and Kd2 have been reported only using spectral-resolution techniques such as native mass spectrometry and slow-exchange NMR. No physical-separation method has yet been shown to yield individual stepwise dissociation constants. Here, we present a nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM)-based approach that physically resolves and quantifies stoichiometric complexes formed at equilibrium. A protein and its ligand are pre-equilibrated in solution, and the resulting complexes of different stoichiometries are separated from one another and from unbound ligand based on differences in electrophoretic mobility. Quantitative peak analysis yields equilibrium fractions of each species, providing the step-resolved thermodynamic data needed for mechanistic insight into cooperative binding. Fitting these data to a stepwise binding model allows direct extraction of individual dissociation constants (Kd1, Kd2, …), whose relative magnitudes reveal the presence and extent of cooperativity. As proof of concept, we applied this method to study the interaction between C-reactive protein (CRP), a homopentameric immune receptor, and a slow off-rate modified aptamer (SOMAmer). The electropherograms resolved and quantified free ligand, 1:1, and 1:2 CRP–SOMAmer complexes, yielding Kd1 = 5.4 ± 1.2 nM and Kd2 = 101 ± 30 nM, which implies strong negative cooperativity. Complexes of higher stoichiometry appeared too weak to form under the investigated concentrations. This study establishes NECEEM as a solution-phase method capable of physically separating and quantifying binding complexes of varying stoichiometries, enabling mechanistic analysis of cooperativity in multivalent systems.