Department of Chemistry and Alberta Glycomics Centre, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
J Am Chem Soc. 2012 Oct 10;134(40):16586-96. doi: 10.1021/ja305213z. Epub 2012 Oct 1.
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 (Ω) measured for protonated gaseous ions of free and ligand-bound truncated (residues 13-139) wild-type (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.
描述了首例详细研究生物素（B）与四聚体蛋白复合物链霉亲和素（S(4)）之间模型高亲和力蛋白-配体相互作用的结构和动力学稳定性的结果。测量了游离和配体结合的截断（残基 13-139）野生型（WT）链霉亲和素，即 S(4)(n+)和(S(4)+4B)(n+)的质子化气态离子的碰撞截面（Ω），在电荷态 n = 12-16 时，发现与电荷态无关，并且与报告的 S(4)和(S(4)+4B)晶体结构的估计值一致（在 10%以内）。这些结果表明，在通过电喷雾电离将复合物从溶液转移到气相时，不会发生显著的结构变化。测量了从质子化（S(4)+4B)(n+)离子中失去 B 的温度依赖性速率常数。在所研究的温度范围内，动力学稳定性随着电荷态从 n = 16 到 13 的降低而增加，但对于 n = 12 和 13 则无法区分。与由 WT 链霉亲和素和五个结合位点突变体（Trp79Phe、Trp108Phe、Trp120Phe、Ser27Ala 和 Tyr43Ala）组成的（S(4)+4B)(13+)离子测量的 B 失去的活化能（E(a)）进行比较表明，至少一些特定的分子间相互作用在气相中得到了保留。对具有不同电荷构型的 WT(S(4)+4B)(12+)离子进行分子动力学模拟的结果支持了这一结论。这项研究的最重要发现是，由于 B 损失的 E(a)（高达 13 kcal/mol）高得多，气态 WT(S(4)+4B)(n+)离子（n = 12-14）在 25°C 时在动力学上比水相中性溶液中的（S(4)+4B)复合物稳定得多。气态（S(4)+4B)(n+)离子和溶剂化（S(4)+4B)复合物之间测量的 E(a)值的差异可以很大程度上归因于迟滞的解离过渡态以及 B 和蛋白质结合腔在溶液中的再水合。
