Hybrid Thermophilic/Mesophilic Enzymes Reveal a Role for Conformational Disorder in Regulation of Bacterial Enzyme I.
Hybrid Thermophilic/Mesophilic Enzymes Reveal a Role for Conformational Disorder in Regulation of Bacterial Enzyme I.
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DOI：
10.1016/j.jmb.2020.05.024
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发表时间：
2020-07-24
影响因子：
5.6
通讯作者：
Venditti V
中科院分区：
文献类型：
--
作者：
Dotas RR;Nguyen TT;Stewart CE Jr;Ghirlando R;Potoyan DA;Venditti V
构象紊乱正在成为生物聚合物的一个重要特征，调节大量的细胞功能，包括信号传导、相分离和酶催化。在这里，我们结合核磁共振、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，我们通过杂交嗜热和嗜温生物体的 EIC 来设计嵌合蛋白，并表征所得构建体的结构、动力学和生物功能。我们表明，EIC 以活性和非活性构象的混合物形式存在，并且功能调节是通过调节活性和非活性状态之间的热力学平衡来实现的。有趣的是，我们还提出了一种混合的嗜热/嗜温酶，它比野生型嗜热酶更稳定且活性更高，这表明杂交嗜热和嗜温蛋白是设计具有显着低温活性的热稳定酶的有效策略。
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.
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DOI：
10.1107/s090744491003982x
发表时间：
2011-04
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Evans PR
通讯作者：
Evans PR
影响因子：
2.2
作者：
CARVER, JP;RICHARDS, RE
通讯作者：
RICHARDS, RE
影响因子：
14.8
作者：
Doucette, Christopher D.;Schwab, David J.;Wingreen, Ned S.;Rabinowitz, Joshua D.
通讯作者：
Rabinowitz, Joshua D.
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
14.8
作者：
Greenfield, Norma J.
通讯作者：
Greenfield, Norma J.