Allostery pervades macromolecular function and drives cooperative binding of ligands to macromolecules. To decipher the mechanisms of cooperative ligand binding it is necessary to define, at a microscopic level, the thermodynamic consequences of binding of each ligand to its energetically coupled site(s). However, extracting these microscopic constants is difficult for macromolecules with more than two binding constants. This goal is complicated because the observable (e.g., NMR chemical shift changes, fluorescence, enthalpy) can be altered by allostery, thereby distorting its proportionality to populations of states. Because it measures mass, native mass spectrometry (MS) can directly quantify the populations of homo-oligomeric protein species with different numbers of bound ligands, provided the populations are proportional to ion counts and that MS-compatible electrolytes do not alter the overall thermodynamics. These measurements can help decipher allosteric mechanisms by providing unparalleled access to the statistical thermodynamic partition function. We used native MS (nMS) to study the cooperative binding of tryptophan (Trp) to Bacillus stearothermophilus trp RNA-binding attenuation protein (TRAP), a ring-shaped homo-oligomeric protein complex with 11 identical binding sites. Mass spectrometrycompatible solutions did not significantly perturb protein structure and thermodynamics as assessed by ITC and NMR spectroscopy. Populations of Trpn-TRAP11 states were quantified as a function of Trp concentration by native mass spectrometry. Population distributions cannot be explained by a non-cooperative binding model but are well described by a nearest neighbor cooperative model. Non-linear least-squares fitting of the populations to a mechanistic model yielded microscopic thermodynamic constants that define the interactions between neighboring binding sites that result in homotropic cooperativity in Trp binding to TRAP.

中文翻译：

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来自天然质谱滴定的种群分布揭示了环形寡聚蛋白TRAP中的近邻协作性。

别构性贯穿大分子功能并驱动配体与大分子的协同结合。为了解释协同配体结合的机理，有必要在微观水平上定义每个配体与其能量耦合位点结合的热力学后果。然而，对于具有两个以上结合常数的大分子，提取这些微观常数是困难的。这个目标是复杂的，因为可以通过变构改变可观察到的（例如NMR化学位移变化，荧光，焓），从而扭曲其与状态种群的比例。因为它可以测量质量，所以天然质谱（MS）可以直接量化具有不同数量的结合配体的同低聚蛋白种类的种群，前提是总体与离子数成正比，并且与MS兼容的电解质不会改变整体热力学。这些测量可通过提供对统计热力学分配函数的无与伦比的访问来帮助破译变构机制。我们使用天然MS（nMS）来研究色氨酸（Trp）与嗜热脂肪芽孢杆菌trp RNA结合衰减蛋白（TRAP）的协同结合，这是一种具有11个相同结合位点的环状均聚寡聚蛋白复合物。通过ITC和NMR光谱评估，与质谱兼容的溶液不会显着干扰蛋白质结构和热力学。通过天然质谱，将Trpn-TRAP11状态的种群定量为Trp浓度的函数。人口分布不能用非合作约束模型来解释，但是可以用最近邻合作模型很好地描述。群体对机理模型的非线性最小二乘拟合产生了微观热力学常数，该常数定义了相邻结合位点之间的相互作用，从而导致Trp与TRAP结合时的各向同性协同作用。