Facultad de Ciencias, Departamento de Quimica Fisica, Universidad de Granada, Granada, Spain.
PLoS One. 2013 Jul 30;8(7):e70013. doi: 10.1371/journal.pone.0070013. Print 2013.
The impact of disulfide bonds on protein stability goes beyond simple equilibrium thermodynamics effects associated with the conformational entropy of the unfolded state. Indeed, disulfide crosslinks may play a role in the prevention of dysfunctional association and strongly affect the rates of irreversible enzyme inactivation, highly relevant in biotechnological applications. While these kinetic-stability effects remain poorly understood, by analogy with proposed mechanisms for processes of protein aggregation and fibrillogenesis, we propose that they may be determined by the properties of sparsely-populated, partially-unfolded intermediates. Here we report the successful design, on the basis of high temperature molecular-dynamics simulations, of six thermodynamically and kinetically stabilized variants of phytase from Citrobacter braakii (a biotechnologically important enzyme) with one, two or three engineered disulfides. Activity measurements and 3D crystal structure determination demonstrate that the engineered crosslinks do not cause dramatic alterations in the native structure. The inactivation kinetics for all the variants displays a strongly non-Arrhenius temperature dependence, with the time-scale for the irreversible denaturation process reaching a minimum at a given temperature within the range of the denaturation transition. We show this striking feature to be a signature of a key role played by a partially unfolded, intermediate state/ensemble. Energetic and mutational analyses confirm that the intermediate is highly unfolded (akin to a proposed critical intermediate in the misfolding of the prion protein), a result that explains the observed kinetic stabilization. Our results provide a rationale for the kinetic-stability consequences of disulfide-crosslink engineering and an experimental methodology to arrive at energetic/structural descriptions of the sparsely populated and elusive intermediates that play key roles in irreversible protein denaturation.
二硫键对蛋白质稳定性的影响超出了与未折叠状态构象熵相关的简单平衡热力学效应。实际上，二硫键交联可能在防止功能失调的聚集中发挥作用，并强烈影响不可逆酶失活的速率，这在生物技术应用中非常重要。虽然这些动力学稳定性效应仍未得到很好的理解，但通过与蛋白质聚集和纤维形成过程的提出机制进行类比，我们提出它们可能取决于稀疏分布、部分展开的中间产物的性质。在这里，我们报告了在高温分子动力学模拟的基础上，成功设计了六种热稳定和动力学稳定的柠檬酸杆菌植酸酶（一种生物技术上重要的酶）变体，这些变体带有一个、两个或三个工程化的二硫键。活性测量和 3D 晶体结构测定表明，工程化的交联不会导致天然结构的剧烈改变。所有变体的失活动力学都表现出强烈的非 Arrhenius 温度依赖性，不可逆变性过程的时间尺度在变性转变范围内达到给定温度的最小值。我们表明，这种显著特征是部分展开的中间状态/集合体发挥关键作用的特征。能量和突变分析证实，中间产物高度展开（类似于朊病毒蛋白错误折叠中提出的关键中间产物），这一结果解释了观察到的动力学稳定性。我们的结果为二硫键交联工程的动力学稳定性后果提供了依据，并提供了一种实验方法，以获得在不可逆蛋白质变性中起关键作用的稀疏分布和难以捉摸的中间产物的能量/结构描述。
