Changes in stability upon charge reversal and neutralization substitution in staphylococcal nuclease are dominated by favorable electrostatic effects.
Changes in stability upon charge reversal and neutralization substitution in staphylococcal nuclease are dominated by favorable electrostatic effects.
复制标题
DOI：
10.1021/bi0266434
复制
发表时间：
2003-01
期刊：
影响因子：
2.9
通讯作者：
J. M. Schwehm;C. A. Fitch;B. Dang;B. García‐Moreno E.;W. Stites
中科院分区：
文献类型：
--
作者：
J. M. Schwehm;C. A. Fitch;B. Dang;B. García‐Moreno E.;W. Stites
在葡萄球菌核酸酶的22个可电离残基上，发生了逆转或中和表面电荷的单点突变。通过盐酸胍变性得到去折叠自由能。这些数据，结合之前获得的相应丙氨酸突变体的稳定性，明确地表明，对几乎所有所研究的野生型侧链的稳定性的主要贡献是与每个残基的带电基团相关的静电效应。除了少数例外，这些电荷稳定了本征态，在电荷中和时，平均损失了0.5千卡/摩尔的稳定性。当电荷反转时，平均不稳定程度增加一倍。用基于线性Poisson-Boltzmann方程有限差分解的连续统法计算静电自由能，当蛋白质内部的极化率用介电常数20表示时，重现了观察到的能量学。然而，在某些情况下，发现了很大的差异，这为计算的改进提供了可能的领域。特别是，在计算中关于变性状态中没有静电相互作用以及本征状态下动态涨落的能量后果的假设似乎需要进一步探索。
Single site mutations that reverse or neutralize a surface charge were made at 22 ionizable residues in staphylococcal nuclease. Unfolding free energies were obtained by guanidine hydrochloride denaturation. These data, in conjunction with previously obtained stabilities of the corresponding alanine mutants, unequivocally show that the dominant contribution to stability for virtually all of the wild-type side chains examined is the electrostatic effect associated with each residue's charged group. With only a few exceptions, these charges stabilize the native state, with an average loss of 0.5 kcal/mol of stability upon neutralization of a charge. When the charge is reversed, the average destabilization is doubled. Structure-based calculations of electrostatic free energy with the continuum method based on the finite difference solution to the linearized Poisson-Boltzmann equation reproduce the observed energetics when the polarizability in the protein interior is represented with a dielectric constant of 20. However, in some cases, large differences are found, giving insight into possible areas for improvement of the calculations. In particular, it appears that the assumptions made in the calculations about the absence of electrostatic interactions in the denatured state and the energetic consequences of dynamic fluctuations in the native state will have to be further explored.