Functional modulation and directed assembly of an enzyme through designed non-natural post-translation modification.
Functional modulation and directed assembly of an enzyme through designed non-natural post-translation modification.
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DOI：
10.1039/c4sc03900a
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发表时间：
2015-07-15
期刊：
影响因子：
8.4
通讯作者：
Jones DD
中科院分区：
文献类型：
--
作者：
Hartley AM;Zaki AJ;McGarrity AR;Robert-Ansart C;Moskalenko AV;Jones GF;Craciun MF;Russo S;Elliott M;Macdonald JE;Jones DD
设计的叠氮苯掺入结合生物正交点击化学来调节酶活性，或促进其在石墨烯上的稳定组装。翻译后修饰（PTM）调节和补充蛋白质功能。本质上，这种高精度事件需要特定的图案和/或相关的修改机制。为了克服阻碍PTM更广泛使用的固有复杂性，我们利用非天然生物相容性点击化学方法来位点特异性修饰TEM β-内酰胺酶，从而增加新的功能。使用计算机模拟设计TEM β-内酰胺酶变体，其中非天然氨基酸对叠氮基-l-苯丙氨酸（azF）置于功能性战略位置，允许通过利用菌株促进的叠氮化物-炔环加成用炔加合物进行残基特异性修饰。实施了三种设计，使得修饰将：（i）抑制TEM活性（Y105azF）;（ii）恢复由初始突变（P174azF）损害的活性;（iii）促进在原始石墨烯（W165azF）上的组装。具有胺官能度的二苄基环辛炔（DBCO）足以调节酶活性。用DBCO-芘加合物修饰TEMW165azF对活性几乎没有影响，尽管修饰位点接近关键催化残基，但允许酶在石墨烯上定向组装，可能有助于蛋白质门控碳晶体管系统的构建。
Designed phenyl azide incorporation combined with bioorthogonal Click chemistry to regulate enzyme activity, or promote its stable assembly on graphene. Post-translational modification (PTM) modulates and supplements protein functionality. In nature this high precision event requires specific motifs and/or associated modification machinery. To overcome the inherent complexity that hinders PTM's wider use, we have utilized a non-native biocompatible Click chemistry approach to site-specifically modify TEM β-lactamase that adds new functionality. In silico modelling was used to design TEM β-lactamase variants with the non-natural amino acid p-azido-l-phenylalanine (azF) placed at functionally strategic positions permitting residue-specific modification with alkyne adducts by exploiting strain-promoted azide–alkyne cycloaddition. Three designs were implemented so that the modification would: (i) inhibit TEM activity (Y105azF); (ii) restore activity compromised by the initial mutation (P174azF); (iii) facilitate assembly on pristine graphene (W165azF). A dibenzylcyclooctyne (DBCO) with amine functionality was enough to modulate enzymatic activity. Modification of TEMW165azF with a DBCO–pyrene adduct had little effect on activity despite the modification site being close to a key catalytic residue but allowed directed assembly of the enzyme on graphene, potentially facilitating the construction of protein-gated carbon transistor systems.