Kinetic Stability of the Streptavidin-Biotin Interaction Enhanced in the Gas Phase
Kinetic Stability of the Streptavidin-Biotin Interaction Enhanced in the Gas Phase
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DOI：
10.1021/ja305213z
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发表时间：
2012-10-10
影响因子：
15
通讯作者：
Klassen, John S.
中科院分区：
文献类型：
--
作者：
Deng, Lu;Broom, Aron;Klassen, John S.
描述了生物素 (B) 和同源四聚蛋白复合物链霉亲和素 (S-4) 之间在气相中的高亲和力蛋白配体相互作用模型的结构和动力学稳定性的首次详细研究结果。测量游离和配体结合的截短（残基 13-139）野生型 (WT) 链霉亲和素（即 S-4(n+) 和 (S-4+4B)(n+)）在电荷态 n = 12-16 时的质子化气态离子的碰撞截面 (Omega)，发现与电荷态无关，并且与报告的晶体结构估计值一致（10% 以内）。 S-4和(S-4+4B)。这些结果表明，通过电喷雾电离将配合物从溶液转移到气相时，不会发生显着的结构变化。测量质子化 (S-4+4B)(n+) 离子中 B 损失的温度依赖性速率常数。在研究的温度范围内，动力学稳定性随着电荷态的降低而增加，从 n = 16 到 13，但对于 n = 12 和 13 来说是无法区分的。比较了由 WT 链霉亲和素和五个结合位点突变体（Trp79Phe、Trp108Phe、 Trp120Phe、Ser27Ala 和 Tyr43Ala）表明至少一些特定的分子间相互作用在气相中得以保留。对不同电荷构型的WT(S-4+4B)(12+)离子进行的分子动力学模拟结果支持了这一结论。这项研究最重要的发现是，n = 12-14 时的气态 WT (S-4+4B)(n+) 离子，由于 B 的损失而具有更大的 E-a（高达 13 kcal mol(-1)），因此在 25℃ 时比中性水溶液中的 (S-4+4B) 络合物动力学更加稳定。气态 (S-4+4B)(n+) 离子和溶剂化 (S-4+4B) 复合物测量的 E-a 值的差异很大程度上可以归因于晚期解离过渡态以及 B 的再水化和溶液中的蛋白质结合腔。
Results of the first detailed study of the structure and kinetic stability of the model high-affinity protein ligand interaction between biotin (B) and the homotetrameric protein complex streptavidin (S-4) in the gas phase are described. Collision cross sections (Omega) measured for protonated gaseous ions of free and ligand-bound truncated (residues 13-139) wildtype (WT) streptavidin, i.e., S-4(n+) and (S-4+4B)(n+) at charge states n = 12-16, were found to be independent of charge state and in agreement (within 10%) with values estimated for crystal structures reported for S-4 and (S-4+4B). These results suggest that significant structural changes do not occur upon transfer of the complexes from solution to the gas phase by electrospray ionization. Temperature-dependent rate constants were measured for the loss of B from the protonated (S-4+4B)(n+) ions. Over the temperature range investigated, the kinetic stability increases with decreasing charge state, from n = 16 to 13, but is indistinguishable for n = 12 and 13. A comparison of the activation energies (E-a) measured for the loss of B from the (S-4+4B)(13+) ions composed of WT streptavidin and five binding site mutants (Trp79Phe, Trp108Phe, Trp120Phe, Ser27Ala, and Tyr43Ala) suggests that at least some of the specific intermolecular interactions are preserved in the gas phase. The results of molecular dynamics simulations performed on WT (S-4+4B)(12+) ions with different charge configurations support this conclusion. The most significant finding of this study is that the gaseous WT (S-4+4B)(n+) ions at n = 12-14, owing to a much larger E-a (by as much as 13 kcal mol(-1)) for the loss of B, are dramatically more stable kinetically at 25 C than the (S-4+4B) complex in aqueous neutral solution. The differences in E-a values measured for the gaseous (S-4+4B)(n+) ions and solvated (S-4+4B) complex can be largely accounted for by a late dissociative transition state and the rehydration of B and the protein binding cavity in solution.