Le Quan Duc, Le An T H, Wang Tong Ye, Krylova Svetlana M, Krylov Sergey N
Department of Chemistry, York University, 4700 Keele St, Toronto, ON M3J 1P3, Canada.
Centre for Research on Biomolecular Interactions, York University, 4700 Keele St, Toronto, ON M3J 1P3, Canada.
Anal Chem. 2025 Sep 2;97(34):18847-18853. doi: 10.1021/acs.analchem.5c03911. Epub 2025 Aug 18.
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 (, ,...). 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 , , 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 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: = 3.0-7.8 nM and = 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 . This study establishes NECEEM as the first solution-phase physical-separation method capable of directly quantifying stepwise affinities and dissecting cooperativity in multivalent systems.
理解协同结合对于表征多聚体蛋白与其配体之间的相互作用至关重要，因为生物学功能通常取决于结合化学计量或变构调节。反过来，对协同性的详细表征需要确定每个结合步骤的平衡解离常数（(K_1)、(K_2)、...）。然而，大多数实验方法依赖于无法分辨共存复合物的总体平均信号，这使得逐步常数只能通过依赖模型的拟合来推断，而仅靠总体数据无法验证这些拟合。迄今为止，只有通过诸如天然质谱和慢交换核磁共振等光谱分辨率技术才能直接（与模型无关）测定这些常数；尚无物理分离方法能够得出(K_1)、(K_2)等。在此，我们提出一种基于平衡混合物非平衡毛细管电泳（NECEEM）的方法，该方法能够物理分离并定量平衡状态下形成的化学计量复合物。将一种蛋白质及其配体在溶液中预先平衡，然后根据不同化学计量的复合物及其游离配体的电泳迁移率将它们彼此分离。定量峰分析可得出每种物质的平衡分数，从而提供逐步解析的热力学数据，从中可直接获得各个(K)值，而无需进行全局模型拟合；它们的相对大小揭示了协同性的存在和程度。作为概念验证，我们研究了先天性免疫系统的同源五聚体急性期蛋白C反应蛋白（CRP）与一种慢解离修饰适体（SOMAmer）之间的相互作用。电泳图分离并定量了游离配体以及1:1和2:1的SOMAmer-CRP复合物，从而能够确定前两个解离常数的95%准确置信区间（ACI）：(K_1 = 3.0 - 7.8 nM)和(K_2 = 41 - 160 nM)，这与强负协同性一致。在高配体与靶标比例下，还检测到了3:1的SOMAmer-CRP复合物，但其峰无法与基线分离，因此无法可靠地测定(K_3)。本研究确立了NECEEM作为第一种能够直接定量多价体系中逐步亲和力并剖析协同性的溶液相物理分离方法。
