Consensus-based protein engineering strategy has been applied to various proteins and it can lead to the design of proteins with enhanced biological performance. Histone-like HUs comprise a protein family with sequence variety within a highly conserved 3D-fold. HU function includes compacting and regulating bacterial DNA in a wide range of biological conditions in bacteria. To explore the possible impact of consensus-based design in the thermodynamic stability of HU proteins, the approach was applied using a dataset of sequences derived from a group of 40 mesostable, thermostable, and hyperthermostable HUs. The consensus-derived HU protein was named HUBest, since it is expected to perform best. The synthetic HU gene was overexpressed in E. coli and the recombinant protein was purified. Subsequently, HUBest was characterized concerning its correct folding and thermodynamic stability, as well as its ability to interact with plasmid DNA. A substantial increase in HUBest stability at high temperatures is observed. HUBest has significantly improved biological performance at ambience temperature, presenting very low K_d values for binding plasmid DNA as indicated from the Gibbs energy profile of HUBest. This K_d may be associated to conformational changes leading to decreased thermodynamic stability and, therefore, higher flexibility at ambient temperature.

中文翻译：

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对热稳定的组蛋白样细菌蛋白HUs进行共识性蛋白质工程改造，可以显着提高稳定性和DNA结合亲和力。

基于共识的蛋白质工程策略已应用于各种蛋白质，并且可以导致具有增强的生物学性能的蛋白质设计。组蛋白样HUs包含高度保守的3D折叠中具有序列多样性的蛋白质家族。HU功能包括在细菌的多种生物学条件下压实和调节细菌DNA。为了探索基于共识的设计对HU蛋白质热力学稳定性的可能影响，该方法使用了来自40个易变，热稳定和超热HU的序列数据集。共识衍生的HU蛋白被称为HU Best，因为它有望表现最佳。合成的HU基因在大肠杆菌中过表达并纯化了重组蛋白。随后，对HU Best进行了表征，涉及其正确的折叠和热力学稳定性以及与质粒DNA相互作用的能力。观察到HU的大量增加高温下的最佳稳定性。HU Best在环境温度下具有显着改善的生物学性能，结合HU Best的Gibbs能量谱显示，结合质粒DNA的K _d值非常低。该K _d可能与构象变化有关，导致热力学稳定性降低，因此在环境温度下具有更高的柔韧性。