The folding unit of phosphofructokinase-2 as defined by the biophysical properties of a monomeric mutant.
The folding unit of phosphofructokinase-2 as defined by the biophysical properties of a monomeric mutant.
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由单体突变体的生物物理特性定义的磷酸果糖激酶-2 的折叠单位。
DOI：
10.1016/j.bpj.2015.04.001
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发表时间：
2015
影响因子：
3.4
通讯作者：
Guixé,Victoria
中科院分区：
文献类型：
--
作者：
Ramírez-Sarmiento,CésarA;Baez,Mauricio;Zamora,RicardoA;Balasubramaniam,Deepa;Babul,Jorge;Komives,ElizabethA;Guixé,Victoria
大肠杆菌磷酸果糖激酶-2（Pfk-2）是一种专性同源二聚体，遵循高度合作的三态折叠机制N2 Particip 2 I Particip 2 U。解离和去折叠之间的强耦合是其界面的结构特征的结果：通过将每个亚基的小结构域缠绕成扁平的β-桶而形成的双分子结构域。虽然通过改变环境（改变温度、加入离液剂）观察到大肠杆菌iPfk-2的分离单体，但在天然条件下未获得分离亚基。基于对结合后自由能变化和局部能量挫折的计算机估计，我们设计了单点突变体以使Pfk-2的界面不稳定。该突变体L93 A在蛋白质浓度低于30μM时是一种无活性单体，通过分析超离心、动态光散射、分子排阻色谱、小角X射线散射和酶动力学测定。活性二聚体的形成可以通过增加蛋白质浓度和通过添加其底物果糖-6-磷酸来诱导。化学和热展开的L93 A单体，然后由圆二色性和动态光散射表明，它展开非合作和孤立的亚基是部分非结构化和边缘稳定。通过高分辨率氢/氘交换质谱确定L93 A单体和F6 P诱导的二聚体的详细结构特征。我们的结果表明，除残基240-309外，分离的亚基比天然二聚体具有总体更高的溶剂可及性。这些残基对应于大多数β-弯曲模块，并显示出与天然二聚体相同的氘摄取程度。我们的研究结果支持Pfk-2的分离单体的疏水核心在天然条件下是溶剂渗透的，并且β-弯曲模块不受单体化突变的影响。
Escherichia coliphosphofructokinase-2 (Pfk-2) is an obligate homodimer that follows a highly cooperative three-state folding mechanism N2↔ 2I ↔ 2U. The strong coupling between dissociation and unfolding is a consequence of the structural features of its interface: a bimolecular domain formed by intertwining of the small domain of each subunit into a flattenedβ-barrel. Although isolated monomers ofE. coliPfk-2 have been observed by modification of the environment (changes in temperature, addition of chaotropic agents), no isolated subunits in native conditions have been obtained. Based on in silico estimations of the change in free energy and the local energetic frustration upon binding, we engineered a single-point mutant to destabilize the interface of Pfk-2. This mutant, L93A, is an inactive monomer at protein concentrations below 30μM, as determined by analytical ultracentrifugation, dynamic light scattering, size exclusion chromatography, small-angle x-ray scattering, and enzyme kinetics. Active dimer formation can be induced by increasing the protein concentration and by addition of its substrate fructose-6-phosphate. Chemical and thermal unfolding of the L93A monomer followed by circular dichroism and dynamic light scattering suggest that it unfolds noncooperatively and that the isolated subunit is partially unstructured and marginally stable. The detailed structural features of the L93A monomer and the F6P-induced dimer were ascertained by high-resolution hydrogen/deuterium exchange mass spectrometry. Our results show that the isolated subunit has overall higher solvent accessibility than the native dimer, with the exception of residues 240–309. These residues correspond to most of theβ-meander module and show the same extent of deuterium uptake as the native dimer. Our results support the idea that the hydrophobic core of the isolated monomer of Pfk-2 is solvent-penetrated in native conditions and that theβ-meander module is not affected by monomerizing mutations.