Asp79 makes a large, unfavorable contribution to the stability of RNase Sa.
Asp79 makes a large, unfavorable contribution to the stability of RNase Sa.
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Asp79 对 RNase Sa 的稳定性做出了巨大但不利的贡献。
DOI：
10.1016/j.jmb.2005.09.091
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发表时间：
2005
期刊：
影响因子：
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通讯作者：
Pace,CNick
中科院分区：
文献类型：
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作者：
Trevino,SaulR;Gokulan,Kuppan;Newsom,Stephanie;Thurlkill,RichardL;Shaw,KevinL;Mitkevich,VladimirA;Makarov,AlexanderA;Sacchettini,JamesC;Scholtz,JMartin;Pace,CNick
核糖核酸酶Sa （RNase Sa）中埋藏最多的两个羧基是Asp33（埋藏99%，pK 2.4）和Asp79（埋藏85%，pK 7.4）。在这些pK值以上，D33A突变体的稳定性比野生型RNase Sa低6kcal/mol， D79A突变体的稳定性比野生型RNase Sa高3.3kcal/mol。这两个羧基在结构上的关键区别在于Asp33形成了3个分子内氢键，而Asp79没有形成分子内氢键。在这里，我们将重点放在Asp79上，并描述了11个Asp79变异的研究。大多数变异比野生型RNase Sa稳定至少2kcal/mol，最有趣的是D79F。在pH为3时，低于Asp79的pK， RNase Sa比D79F变体稳定0.3kcal/mol。在pH 8.5，高于Asp79的pK时，RNase Sa的稳定性比D79F变体低3.7kcal/mol。Asp79对稳定性的不利贡献似乎是由于埋藏电荷的玻恩自能，更重要的是由于不利的电荷-电荷相互作用。为了抵消负电荷对Asp79的影响，我们制备了Q94K突变体，晶体结构表明，Lys的氨基与Asp79的羧基形成了一个氢键离子对（距离，2.71Å；角度，100°）。Q94K变体在pH值为3时的稳定性与野生型基本相同，其中Asp79不带电，但在pH值为8.5时，其稳定性比野生型RNase Sa高1kcal/mol，其中Asp79带电。在疏水性、空间应变、玻恩自能和静电相互作用方面的差异似乎都有助于在变体中观察到的稳定性范围。在可能的情况下，用非极性侧链代替埋藏的、非氢键的、可电离的侧链是提高蛋白质稳定性的一种极好的方法。
The two most buried carboxyl groups in ribonuclease Sa (RNase Sa) are Asp33 (99% buried; pK 2.4) and Asp79 (85% buried; pK 7.4). Above these pK values, the stability of the D33A variant is 6kcal/mol less than wild-type RNase Sa, and the stability of the D79A variant is 3.3kcal/mol greater than wild-type RNase Sa. The key structural difference between the carboxyl groups is that Asp33 forms three intramolecular hydrogen bonds, and Asp79 forms no intramolecular hydrogen bond. Here, we focus on Asp79 and describe studies of 11 Asp79 variants. Most of the variants were at least 2kcal/mol more stable than wild-type RNase Sa, and the most interesting was D79F. At pH 3, below the pK of Asp79, RNase Sa is 0.3kcal/mol more stable than the D79F variant. At pH 8.5, above the pK of Asp79, RNase Sa is 3.7kcal/mol less stable than the D79F variant. The unfavorable contribution of Asp79 to the stability appears to result from the Born self-energy of burying the charge and, more importantly, from unfavorable charge–charge interactions. To counteract the effect of the negative charge on Asp79, we prepared the Q94K variant and the crystal structure showed that the amino group of the Lys formed a hydrogen-bonded ion pair (distance, 2.71Å; angle, 100°) with the carboxyl group of Asp79. The stability of the Q94K variant was about the same as the wild-type at pH 3, where Asp79 is uncharged, but 1kcal/mol greater than that of wild-type RNase Sa at pH 8.5, where Asp79 is charged. Differences in hydrophobicity, steric strain, Born self-energy, and electrostatic interactions all appear to contribute to the range of stabilities observed in the variants. When it is possible, replacing buried, non-hydrogen bonded, ionizable side-chains with non-polar side-chains is an excellent means of increasing protein stability.