Backbone Dynamics and Global Effects of an Activating Mutation in Minimized Mtu RecA Inteins
Backbone Dynamics and Global Effects of an Activating Mutation in Minimized Mtu RecA Inteins
复制标题
最小化 Mtu RecA 内含肽激活突变的主链动力学和全局效应
DOI：
10.1016/j.jmb.2010.05.044
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发表时间：
2010-07-23
影响因子：
5.6
通讯作者：
Wang, Chunyu
中科院分区：
文献类型：
--
作者：
Du, Zhenming;Liu, Yangzhong;Wang, Chunyu
内含肽介导蛋白质剪接，在生物技术和蛋白质工程中有许多应用。单个缬氨酸到亮氨酸的突变 (V67L) 可以整体增强最小化结核分枝杆菌 RecA 内含肽中的剪接和相关切割反应。然而，V67L突变导致晶体结构变化很小。为了测试蛋白质动力学是否有助于 V67L 突变的活性增强，我们通过溶液 NMR 研究了最小化和工程化的内含肽 Delta Delta Ihh-V67CM 和单个 V67L 突变体 Delta Delta Ihh-L67CM 的构象和动力学。化学位移扰动证实 V67L 突变会引起整体变化，包括内含肽 N 末端和 C 末端的变化，这些是蛋白质剪接的活性位点。单个 V67L 突变显着降低了全球氢交换速率，表明向更稳定构象的转变以及整体分布的减少。虽然 V67L 突变对皮秒到纳秒时间尺度上的运动几乎没有造成变化，但微秒到毫秒时间尺度上的运动影响涉及保守 F 区组氨酸和 C 末端天冬酰胺的区域，这些残基对 C 末端裂解很重要。 V67L 突变被认为是通过减少内含肽结构的整体分布和修改活性位点来激活剪接。 (C) 2010 Elsevier Ltd. 保留所有权利。
Inteins mediate protein splicing, which has found many applications in biotechnology and protein engineering. A single valine-to-leucine mutation (V67L) can globally enhance splicing and related cleavage reactions in minimized Mycobacterium tuberculosis RecA inteins. However, V67L mutation causes little change in crystal structures. To test whether protein dynamics contribute to activity enhancement in the V67L mutation, we have studied the conformations and dynamics of the minimized and engineered intein Delta Delta Ihh-V67CM and a single V67L mutant, Delta Delta Ihh-L67CM, by solution NMR. Chemical shift perturbations established that the V67L mutation causes global changes, including changes at the N-terminus and C-terminus of the intein, which are active sites for protein splicing. The single V67L mutation significantly slows hydrogen-exchange rates globally, indicating a shift to more stable conformations and reduction in ensemble distribution. Whereas the V67L mutation causes little change for motions on the picosecond-to-nanosecond timescale, motions on the microsecond-to-millisecond timescale affect a region involving the conserved F-block histidine and C-terminal asparagine, which are residues important for C-terminal cleavage. The V67L mutation is proposed to activate splicing by reducing the ensemble distribution of the intein structure and by modifying the active sites. (C) 2010 Elsevier Ltd. All rights reserved.