Networks of electrostatic and hydrophobic interactions modulate the complex folding free energy surface of a designed βα protein
Networks of electrostatic and hydrophobic interactions modulate the complex folding free energy surface of a designed βα protein
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DOI：
10.1073/pnas.1818744116
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发表时间：
2019-04-02
影响因子：
11.1
通讯作者：
Matthews, C. Robert
中科院分区：
文献类型：
--
作者：
Basak, Sujit;Nobrega, R. Paul;Matthews, C. Robert
蛋白质的成功从头设计可以提供对稳定性的物理化学基础、进化在限制氨基酸序列中的作用以及为工程应用生产可定制平台的见解。先前设计的天然存在的β α折叠Di-III_14的盐酸胍（GdnHCl;离子变性剂）实验揭示了协同的两态解折叠转变和适度的稳定性。在我们的实验室中的连续流混合实验揭示了一个简单的两态反应在微秒到毫秒的时间范围内，并符合热力学结果。与此形成鲜明对比的是，蛋白质在中性变性剂尿素中仍折叠至9.25 M，并且在水中的氢交换（HDX）NMR分析显示存在许多高能状态，这些状态在大于秒的时间尺度上相互转换。HDX的复杂保护模式与表面上的一对静电网络和蛋白质内部的疏水侧链的广泛网络密切对应。突变分析表明，静电和疏水网络有助于WT蛋白对尿素变性的抗性;值得注意的是，蛋白表面上的单电荷反转恢复了预期的尿素敏感性。能量表面的粗糙度反映了密集堆积的疏水核心;仅去除两个甲基基团就消除了高能态并产生了光滑的表面。一个非常稳定的β-α折叠包含静电和疏水网络的设计创造了一个复杂的能量表面，在天然蛋白质中很少观察到。
The successful de novo design of proteins can provide insights into the physical chemical basis of stability, the role of evolution in constraining amino acid sequences, and the production of customizable platforms for engineering applications. Previous guanidine hydrochloride (GdnHCl; an ionic denaturant) experiments of a designed, naturally occurring beta alpha fold, Di-III_14, revealed a cooperative, two-state unfolding transition and a modest stability. Continuous-flow mixing experiments in our laboratory revealed a simple two-state reaction in the microsecond to millisecond time range and consistent with the thermodynamic results. In striking contrast, the protein remains folded up to 9.25 M in urea, a neutral denaturant, and hydrogen exchange (HDX) NMR analysis in water revealed the presence of numerous high-energy states that inter-convert on a time scale greater than seconds. The complex protection pattern for HDX corresponds closely with a pair of electrostatic networks on the surface and an extensive network of hydrophobic side chains in the interior of the protein. Mutational analysis showed that electrostatic and hydrophobic networks contribute to the resistance to urea denaturation for the WT protein; remarkably, single charge reversals on the protein surface restore the expected urea sensitivity. The roughness of the energy surface reflects the densely packed hydrophobic core; the removal of only two methyl groups eliminates the high-energy states and creates a smooth surface. The design of a very stable beta alpha fold containing electrostatic and hydrophobic networks has created a complex energy surface rarely observed in natural proteins.