Rational modification of protein stability by the mutation of charged surface residues.
Rational modification of protein stability by the mutation of charged surface residues.
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DOI：
10.1021/bi992091m
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发表时间：
2000-01
期刊：
影响因子：
2.9
通讯作者：
S. Spector;Minghui Wang;S. A. Carp;J. Robblee;Z. Hendsch;R. Fairman;B. Tidor;D. Raleigh
中科院分区：
文献类型：
--
作者：
S. Spector;Minghui Wang;S. A. Carp;J. Robblee;Z. Hendsch;R. Fairman;B. Tidor;D. Raleigh
使用连续体方法计算带电侧链和极性侧链对小型 41 个残基螺旋蛋白（外围亚基结合域）整体稳定性的静电贡献。这些计算的结果表明，相对于疏水性电子等排体，一些残基是不稳定的。选择一个位置来测试这些计算的结果。 Arg8 位于蛋白质表面正静电势区域。计算表明 Arg8 对整体稳定性产生了显着的、不利的静电贡献。本文描述的实验代表了理论方法的首次直接实验测试，利用固相肽合成来合并近似等排的氨基酸取代。 Arg8 被正亮氨酸 (Nle) 取代，正亮氨酸 (Nle) 是一种疏水性且近似等排的氨基酸，或被 α-氨基己二酸 (Aad) 取代，后者也是近似等排但带相反电荷的氨基酸。以这种方式，可以将静电相互作用与疏水性和范德华相互作用的影响隔离开。 Arg8Nle 和 Arg8Aad 都比野生型序列更热稳定，证明了计算的有效性。这些替代导致野生型 T(m) 在 52.6 摄氏度下的稳定性分别增加 0.86 和 1.08 kcal mol(-)(1)。 Arg8Nle 的稳定性特别令人感兴趣，因为用疏水残基取代表面电荷会导致蛋白质稳定性增加的罕见情况。
Continuum methods were used to calculate the electrostatic contributions of charged and polar side chains to the overall stability of a small 41-residue helical protein, the peripheral subunit-binding domain. The results of these calculations suggest several residues that are destabilizing, relative to hydrophobic isosteres. One position was chosen to test the results of these calculations. Arg8 is located on the surface of the protein in a region of positive electrostatic potential. The calculations suggest that Arg8 makes a significant, unfavorable electrostatic contribution to the overall stability. The experiments described in this paper represent the first direct experimental test of the theoretical methods, taking advantage of solid-phase peptide synthesis to incorporate approximately isosteric amino acid substitutions. Arg8 was replaced with norleucine (Nle), an amino acid that is hydrophobic and approximately isosteric, or with alpha-amino adipic acid (Aad), which is also approximately isosteric but oppositely charged. In this manner, it is possible to isolate electrostatic interactions from the effects of hydrophobic and van der Waals interactions. Both Arg8Nle and Arg8Aad are more thermostable than the wild-type sequence, testifying to the validity of the calculations. These replacements led to stability increases at 52.6 degrees C, the T(m) of the wild-type, of 0.86 and 1.08 kcal mol(-)(1), respectively. The stability of Arg8Nle is particularly interesting as a rare case in which replacement of a surface charge with a hydrophobic residue leads to an increase in the stability of the protein.