Thermal versus guanidine-induced unfolding of ubiquitin. An analysis in terms of the contributions from charge-charge interactions to protein stability
Thermal versus guanidine-induced unfolding of ubiquitin. An analysis in terms of the contributions from charge-charge interactions to protein stability
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DOI：
10.1021/bi9905819
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发表时间：
1999-06-22
期刊：
影响因子：
2.9
通讯作者：
Sanchez-Ruiz, JM
中科院分区：
文献类型：
--
作者：
Ibarra-Molero, B;Loladze, VV;Sanchez-Ruiz, JM
我们在荧光和圆二色性测量的基础上，在25℃和酸性pH范围内表征了胍诱导的酵母和牛泛素的展开。根据高胍展开数据，通过线性外推计算得出的展开吉布斯能量变化对ph的依赖性非常弱。对这一结果的一个简单解释涉及以下两个假设：(1)可电离基团的带电原子暴露于天然泛素中的溶剂中（由可达表面积计算支持），与折叠时电荷脱溶相关的吉布斯能量贡献（pK位移的来源）很小；(2)电荷-电荷相互作用（折叠时pK位移的另一个来源）在浓缩氯化胍溶液中被筛选出来。我们还利用差示扫描量热法表征了这两种蛋白质的热展开。从量热数据计算的吉布斯能量变化确实强烈依赖于pH值，我们将这一结果归因于电荷-电荷相互作用的pH依赖性（在没有胍的情况下不会消除）。事实上，我们发现两个系列的实验展开吉布斯能量变化之间的差异（通过线性外推从高胍展开数据和不含胍的热变性数据中确定）与使用溶剂可及性校正的Tanford-Kirkwood模型获得的电荷-电荷相互作用对泛素展开吉布斯能量变化的贡献的理论估计之间的差异非常一致。以及Bashford-Karplus（简化的地点集）近似。这种贡献被发现在中性pH下是稳定的，因为大多数带电基团在nat上；ive蛋白主要与相反电荷的基团相互作用，这一事实，加上没有大的电荷溶解贡献，可能解释了泛素在中性ph下的高稳定性。总的来说，我们的分析表明，通过充分重新设计天然蛋白表面溶剂暴露的带电残基的分布，可以增强蛋白质的热稳定性。
We have characterized the guanidine-induced unfolding of both yeast and bovine ubiquitin at 25 degrees C and in the acidic pH range on the basis of fluorescence and circular dichroism measurements. Unfolding Gibbs energy changes calculated by linear extrapolation from high guanidine unfolding data are found to depend very weakly on pH. A simple explanation for this result involves the two following assumptions: (1) charged atoms of ionizable groups are exposed to the solvent in native ubiquitin (as supported by accessible surface area calculations), and Gibbs energy contributions associated with charge desolvation upon folding (a source of pK shifts) are small; (2) charge-charge interactions (another source of pK shifts upon folding) are screened out in concentrated guanidinium chloride solutions. We have also characterized the thermal unfolding of both proteins using differential scanning calorimetry. Unfolding Gibbs energy changes calculated from calorimetric data do depend strongly on pH, a result that we attribute to the pH dependence of charge-charge interactions (not eliminated in the absence of guanidine). In fact, we find good agreement between the difference between the two series of experimental unfolding Gibbs energy changes (determined from high guanidine unfolding data by linear extrapolation and from thermal denaturation data in the absence of guanidine) and the theoretical estimates of the contribution from charge-charge interactions to the Gibbs energy change for ubiquitin unfolding obtained by using the solvent-accessibility-corrected Tanford-Kirkwood model, together with the Bashford-Karplus (reduced-set-of-sites) approximation. This contribution is found to be stabilizing at neutral pH, because most charged groups on the nat;ive protein interact mainly with groups of the opposite charge, a fact that, together with the absence of large charge-desolvation contributions, may explain the high stability of ubiquitin at neutral pH. In general, our analysis suggests the possibility of enhancing protein thermal stability by adequately redesigning the distribution of solvent-exposed, charged residues on the native protein surface.