Conformational disorder is emerging as an important feature of biopolymers, regulating a vast array of cellular functions, including signaling, phase separation, and enzyme catalysis. Here we combine NMR, crystallography, computer simulations, protein engineering, and functional assays to investigate the role played by conformational heterogeneity in determining the activity of the C-terminal domain of bacterial Enzyme I (EIC). In particular, we design chimeric proteins by hybridizing EIC from thermophilic and mesophilic organisms, and we characterize the resulting constructs for structure, dynamics, and biological function. We show that EIC exists as a mixture of active and inactive conformations and that functional regulation is achieved by tuning the thermodynamic balance between active and inactive states. Interestingly, we also present a hybrid thermophilic/mesophilic enzyme that is thermostable and more active than the wild-type thermophilic enzyme, suggesting that hybridizing thermophilic and mesophilic proteins is a valid strategy to engineer thermostable enzymes with significant low-temperature activity.

构象障碍正在成为生物聚合物的重要特征，它调节着广泛的细胞功能，包括信号传导，相分离和酶催化。在这里，我们结合NMR，晶体学，计算机模拟，蛋白质工程和功能分析，以研究构象异质性在确定细菌酶I（EIC）C端结构域的活性中所起的作用。特别是，我们通过将嗜热和嗜温生物的EIC进行杂交来设计嵌合蛋白，并表征所得结构的结构，动力学和生物学功能。我们表明，EIC作为活性和非活性构象的混合物存在，并且通过调节活性和非活性状态之间的热力学平衡来实现功能调节。有趣的是