Structural and thermodynamic consequences of burial of an artificial ion pair in the hydrophobic interior of a protein
Structural and thermodynamic consequences of burial of an artificial ion pair in the hydrophobic interior of a protein
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DOI：
10.1073/pnas.1402900111
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发表时间：
2014-08-12
影响因子：
11.1
通讯作者：
Garcia-Moreno, Bertrand E.
中科院分区：
文献类型：
--
作者：
Robinson, Aaron C.;Castaneda, Carlos A.;Garcia-Moreno, Bertrand E.
通过进行V23 E和L36 K取代，工程化了埋在葡萄球菌核酸酶的疏水核心中的人工电荷对。单独掩埋时，Glu-23和Lys-36的pK（a）值均接近7。当埋在一起时，它们的pK（a）值似乎是正常的。掩埋的Glu-Lys对的可电离部分相距2.6埃。在pH 7时，它们之间的相互作用值为5 kcal/mol。尽管这种强相互作用，掩埋的谷氨酸-赖氨酸对显着不稳定的蛋白质，因为明显的库仑相互作用是足以抵消只有一个的两个掩埋电荷的脱水。除了轻微的重组的偶极子和水的渗透与相对较高的介电常数报告的掩埋离子对一致，没有证据表明，存在两个电荷的疏水内部的蛋白质诱导任何显着的结构重组。人工离子对在高度疏水环境中的成功工程化表明，在脱水环境中掩埋的Glu-Lys对可以带电，并且可以工程化电荷簇，其松散地类似于具有高热力学稳定性的支架蛋白中的催化位点，而不需要专门的结构适应。
An artificial charge pair buried in the hydrophobic core of staphylococcal nuclease was engineered by making the V23E and L36K substitutions. Buried individually, Glu-23 and Lys-36 both titrate with pK(a) values near 7. When buried together their pK(a) values appear to be normal. The ionizable moieties of the buried Glu-Lys pair are 2.6 angstrom apart. The interaction between them at pH 7 is worth 5 kcal/mol. Despite this strong interaction, the buried Glu-Lys pair destabilizes the protein significantly because the apparent Coulomb interaction is sufficient to offset the dehydration of only one of the two buried charges. Save for minor reorganization of dipoles and water penetration consistent with the relatively high dielectric constant reported by the buried ion pair, there is no evidence that the presence of two charges in the hydrophobic interior of the protein induces any significant structural reorganization. The successful engineering of an artificial ion pair in a highly hydrophobic environment suggests that buried Glu-Lys pairs in dehydrated environments can be charged and that it is possible to engineer charge clusters that loosely resemble catalytic sites in a scaffold protein with high thermodynamic stability, without the need for specialized structural adaptations.