Department of Chemistry, Iowa State University, Ames, IA 50011, USA.
Macromolecular X-ray Crystallography Facility, Office of Biotechnology, Iowa State University, Ames, IA 50011, USA.
J Mol Biol. 2020 Jul 24;432(16):4481-4498. doi: 10.1016/j.jmb.2020.05.024. Epub 2020 Jun 3.
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.
构象无序正成为生物聚合物的一个重要特征，调节着包括信号转导、相分离和酶催化在内的广泛细胞功能。在这里，我们结合 NMR、晶体学、计算机模拟、蛋白质工程和功能测定来研究构象异质性在决定细菌酶 I (EIC) C 端结构域活性中所起的作用。特别是，我们通过杂交来自嗜热和中温生物的 EIC 来设计嵌合蛋白，并对所得构建体的结构、动力学和生物学功能进行了表征。我们表明，EIC 存在于活性和非活性构象的混合物中，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，我们还展示了一种混合的嗜热/中温酶，它比野生型嗜热酶更耐热且更具活性，这表明杂交嗜热和中温蛋白是一种有效的策略，可以设计具有显著低温活性的耐热酶。
