Spector S, Wang M, Carp S A, Robblee J, Hendsch Z S, Fairman R, Tidor B, Raleigh D P
Department of Physiology and Biophysics, State University of New York at Stony Brook, Stony Brook, New York 11794-8661, USA.
Biochemistry. 2000 Feb 8;39(5):872-9. doi: 10.1021/bi992091m.
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.
采用连续介质方法计算了带电和极性侧链对一个由41个残基组成的小型螺旋蛋白（外周亚基结合结构域）整体稳定性的静电贡献。这些计算结果表明，相对于疏水等排体，有几个残基会使稳定性降低。选择了一个位置来检验这些计算结果。精氨酸8（Arg8）位于蛋白质表面带正静电势的区域。计算表明，Arg8对整体稳定性有显著的、不利的静电贡献。本文所述的实验利用固相肽合成来引入近似等排的氨基酸替代物，是对理论方法的首次直接实验验证。将Arg8替换为正亮氨酸（Nle），一种疏水且近似等排的氨基酸，或替换为α-氨基己二酸（Aad），它也近似等排但带相反电荷。通过这种方式，可以将静电相互作用与疏水和范德华相互作用的影响分离开来。Arg8Nle和Arg8Aad都比野生型序列更耐热，这证明了计算的有效性。在野生型的熔点温度52.6℃下，这些替换分别使稳定性提高了0.86和1.08千卡/摩尔。Arg8Nle的稳定性特别有趣，因为这是一个罕见的例子，即用疏水残基取代表面电荷会导致蛋白质稳定性增加。
