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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Use of sequence duplication to engineer a ligand-triggered, long-distance molecular switch in T4 lysozyme.
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Use of sequence duplication to engineer a ligand-triggered, long-distance molecular switch in T4 lysozyme.

机译：使用序列重复工程改造T4溶菌酶中的配体触发的长距离分子开关。

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摘要

We have designed a molecular switch in a T4 lysozyme construct that controls a large-scale translation of a duplicated helix. As shown by crystal structures of the construct with the switch on and off, the conformational change is triggered by the binding of a ligand (guanidinium ion) to a site that in the wild-type protein was occupied by the guanidino head group of an Arg. In the design template, a duplicated helix is flanked by two loop regions of different stabilities. In the "on" state, the N-terminal loop is weakly structured, whereas the C-terminal loop has a well defined conformation that is stabilized by means of nonbonded interactions with the Arg head group. The truncation of the Arg to Ala destabilizes this loop and switches the protein to the "off" state, in which the duplicated helix is translocated approximately 20 A. Guanidinium binding restores the key interactions, restabilizes the C-terminal loop, and restores the "on" state. Thus, the presence of an external ligand, which is unrelated to the catalytic activity of the enzyme, triggers the inserted helix to translate 20 A away from the binding site. The results illustrate a proposed mechanism for protein evolution in which sequence duplication followed by point mutation can lead to the establishment of new function.
机译：我们在T4溶菌酶构建体中设计了一个分子开关，该分子开关可控制重复螺旋的大规模翻译。如通过打开和关闭的构建体的晶体结构所示，构象变化是由配体（胍盐离子）与野生型蛋白质中被Arg的胍基头部占据的位点结合而触发的。在设计模板中，重复的螺旋两侧是两个具有不同稳定性的环区域。在“开”状态下，N末端环的结构较弱，而C末端环具有定义明确的构象，该构象通过与Arg头基的非键相互作用而得以稳定。将Arg截短为Ala会使该环不稳定，并将蛋白质切换为“关闭”状态，在该状态下，重复的螺旋转移到大约20A。胍基结合可恢复关键相互作用，使C末端环恢复稳定，并恢复“开启”状态。因此，与酶的催化活性无关的外部配体的存在触发插入的螺旋将20A翻译成远离结合位点。结果说明了蛋白质进化的一种拟议机制，其中序列复制继之以点突变可导致建立新功能。

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