Department of Biophysics , Johns Hopkins University , Baltimore , Maryland 21218 , United States.
Chemistry Department , U.S. Naval Academy , Annapolis , Maryland 21402 , United States.
J Phys Chem B. 2018 Mar 8;122(9):2516-2524. doi: 10.1021/acs.jpcb.7b12121. Epub 2018 Feb 21.
Thirty years ago, Hwang and Warshel suggested that a microenvironment preorganized to stabilize an ion pair would be incapable of reorganizing to stabilize the reverse ion pair. The implications were that (1) proteins have a limited capacity to reorganize, even under the influence of strong interactions, such as those present when ionizable groups are buried in the hydrophobic interior of a protein, and (2) the inability of proteins to tolerate the reversal of buried ion pairs demonstrates the limitations inherent to continuum electrostatic models of proteins. Previously we showed that when buried individually in the interior of staphylococcal nuclease, Glu23 and Lys36 have p K values near pH 7, but when buried simultaneously, they establish a strong interaction of ∼5 kcal/mol and have p K values shifted toward more normal values. Here, using equilibrium thermodynamic measurements, crystal structures, and NMR spectroscopy experiments, we show that although the reversed, individual substitutions-Lys23 and Glu36-also have p K values near 7, when buried together, they neither establish a strong interaction nor promote reorganization of their microenvironment. These experiments both confirm Warshel's original hypothesis and expand it by showing that it applies to reorganization, as demonstrated by our artificial ion pairs, as well as to preorganization as is commonly argued for motifs that stabilize naturally occurring ion pairs in polar microenvironments. These data constitute a challenging benchmark useful to test the ability of structure-based algorithms to reproduce the compensation between self-energy, Coulomb and polar interactions in hydrophobic environments of proteins.
三十年前，黄和沃歇尔提出，预先组织起来稳定一对离子的微环境将无法重新组织以稳定相反的离子对。这意味着：（1）蛋白质的重组能力有限，即使受到强烈相互作用的影响，例如可离子化基团埋藏在蛋白质的疏水环境中时存在的相互作用；（2）蛋白质无法容忍埋藏的离子对的反转，这表明连续静电蛋白质模型固有的局限性。此前，我们表明，当单个埋藏在葡萄球菌核酸酶的内部时，Glu23 和 Lys36 的 pK 值接近 pH 7，但当同时埋藏时，它们建立了约 5 千卡/摩尔的强相互作用，并且 pK 值向更正常的值移动。在这里，我们使用平衡热力学测量、晶体结构和 NMR 光谱实验表明，尽管反向的单个取代 Lys23 和 Glu36 也具有接近 7 的 pK 值，但当一起埋藏时，它们既没有建立强相互作用，也没有促进其微环境的重组。这些实验既证实了沃歇尔的原始假设，又通过展示其适用于重组，如我们的人工离子对所示，以及适用于通常被认为稳定极性微环境中天然离子对的基序的预组织，扩展了该假设。这些数据构成了一个具有挑战性的基准，可用于测试基于结构的算法在蛋白质疏水环境中复制自能、库仑和极性相互作用之间补偿的能力。
