Nonequilibrium Capillary Electrophoresis of Equilibrium Mixtures (NECEEM)-Enabled Mechanistic Analysis of Cooperative Binding: Application to C-Reactive Protein–SOMAmer Complexes

Understanding cooperative binding is essential for characterizing interactions between multimeric proteins and their ligands, because biological function often depends on binding stoichiometry or allosteric regulation. Detailed characterization of cooperativity, in turn, requires determination of the equilibrium dissociation constants for each binding step (<i>K</i>_d1, <i>K</i>_d2,...). However, most experimental methods rely on ensemble-averaged signals that cannot resolve coexisting complexes, forcing stepwise constants to be inferred from model-dependent fits that cannot be validated with ensemble data alone. To date, direct (model-independent) determinations of these constants have been reported only with spectral-resolution techniques such as native MS and slow-exchange NMR; no physical-separation method has yet delivered <i>K</i>_d1, <i>K</i>_d2, etc. 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 free ligand according to their electrophoretic mobilities. Quantitative peak analysis yields the equilibrium fractions of each species, providing step-resolved thermodynamic data from which individual <i>K</i>_d values are obtained directly, without global model fitting; their relative magnitudes reveal the presence and extent of cooperativity. As proof of concept, we studied the interaction between C-reactive protein (CRP), a homopentameric acute-phase protein of the innate immune system, and a slow off-rate modified aptamer (SOMAmer). The electropherograms resolved and quantified free ligand as well as 1:1 and 2:1 SOMAmer-CRP complexes, allowing determination of the 95% accuracy confidence intervals (ACI) for the first two dissociation constants: <i>K</i>_d1 = 3.0-7.8 nM and <i>K</i>_d2 = 41-160 nM, consistent with strong negative cooperativity. At high ligand-to-target ratios, 3:1 SOMAmer-CRP complex was also detected, but its peak could not be baseline-resolved, precluding reliable determination of <i>K</i>_d3. This study establishes NECEEM as the first solution-phase physical-separation method capable of directly quantifying stepwise affinities and dissecting cooperativity in multivalent systems.