Population Distributions from Native Mass Spectrometry Titrations Reveal Nearest-Neighbor Cooperativity in the Ring-Shaped Oligomeric Protein TRAP.,Biochemistry

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Population Distributions from Native Mass Spectrometry Titrations Reveal Nearest-Neighbor Cooperativity in the Ring-Shaped Oligomeric Protein TRAP.
Biochemistry ( IF 2.9 ) Pub Date : 2020-06-19 , DOI: 10.1021/acs.biochem.0c00352
Melody L Holmquist ₁ , Elihu C Ihms ₂ , Paul Gollnick ₃ , Vicki H Wysocki _{1,

4} , Mark P Foster ₁

Affiliation

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 sites, because the observable [e.g., nuclear magnetic resonance (NMR) chemical shift changes, fluorescence, and enthalpy] can be altered by allostery, thereby distorting its proportionality to site occupancy. 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 stearothermophilustrp RNA binding attenuation protein (TRAP), a ring-shaped homo-oligomeric protein complex with 11 identical binding sites. MS-compatible solutions did not significantly perturb protein structure or thermodynamics as assessed by isothermal titration calorimetry and NMR spectroscopy. Populations of Trp_n-TRAP₁₁ states were quantified as a function of Trp concentration by nMS. The population distributions could not be explained by a noncooperative binding model but were described well by a mechanistic nearest-neighbor cooperative model. Nonlinear least-squares fitting yielded microscopic thermodynamic constants that define the interactions between neighboring binding sites. This approach may be applied to quantify thermodynamic cooperativity in other ring-shaped proteins.

中文翻译：

天然质谱滴定的群体分布揭示了环形寡聚蛋白 TRAP 中的最近邻协同性。

变构遍及大分子功能并驱动配体与大分子的协同结合。为了破译协同配体结合的机制，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位点的大分子来说，提取这些微观常数是困难的，因为可观察到的[例如核磁共振（NMR）化学位移变化、荧光和焓]可以通过变构改变，从而扭曲其与位点的比例占用。天然质谱 (MS) 可以直接量化具有不同数量结合配体的同源寡聚蛋白质种类的数量，前提是这些数量与离子计数成正比，并且 MS 兼容的电解质不会改变整体热力学。这些测量可以通过提供无与伦比的统计热力学配分函数来帮助破译变构机制。我们使用天然 MS (nMS) 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，TRAP 是一种具有 11 个相同结合位点的环形同源寡聚蛋白复合物。通过等温滴定量热法和核磁共振波谱评估，MS 兼容溶液不会显着扰乱蛋白质结构或热力学。通过 nMS 将 Trp _n -TRAP ₁₁状态的数量量化为 Trp 浓度的函数。种群分布不能用非合作结合模型来解释，但可以用机械最近邻合作模型很好地描述。非线性最小二乘拟合产生了定义相邻结合位点之间相互作用的微观热力学常数。这种方法可用于量化其他环状蛋白质的热力学协同性。

更新日期：2020-07-14

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