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One-Pot, Photocontrolled Enzymatic Assembly of the Structure-Defined Heterotypic Polyubiquitin Chain
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2023-05-18 , DOI: 10.1021/jacs.3c01912
Takafumi Furuhata 1 , Phebee Angeline Devadasan Racheal 2 , Iori Murayama 1 , Usano Toyoda 1 , Akimitsu Okamoto 1, 2
Affiliation  

Heterotypic polyubiquitins are an emerging class of polyubiquitins that have attracted interest because of their potential diversity of structures and physiological functions. There is an increasing demand for structure-defined synthesis of heterotypic chains to investigate the topological factors underlying the intracellular signals that are characteristically mediated by the heterotypic chain. However, the applicability of chemical and enzymatic polyubiquitin synthesis developed to date has been limited by laborious rounds of ligation and purification or by a lack of modularity of the chain structure with respect to the length and the branch position. Here, we established a one-pot, photocontrolled synthesis of structurally defined heterotypic polyubiquitin chains. We designed ubiquitin derivatives with a photolabile protecting group at a lysine residue used for polymerization. Repetitive cycles of linkage-specific enzymatic elongation and photoinduced deprotection of the protected ubiquitin units enabled stepwise addition of ubiquitins with appropriate functionalities to control the length and branching positions. The positional control of branching was achieved without isolation of intermediates, allowing one-pot synthesis of K63 triubiqutin chains and a K63/K48 heterotypic tetraubiquitin chain with defined branching positions. The present study provides a chemical platform for the efficient construction of long polyubiquitin chains with defined branch structures that will facilitate the understanding of the essential relationships between functions and structures of the heterotypic chain that have hitherto been overlooked.

中文翻译:

一锅法,结构定义的异型多聚泛素链的光控酶组装

异型多聚泛素是一类新兴的多聚泛素,由于其结构和生理功能的潜在多样性而引起了人们的兴趣。对异型链的结构定义合成的需求越来越大,以研究由异型链介导的细胞内信号的拓扑因素。然而,迄今为止开发的化学和酶促多聚泛素合成的适用性受到费力的连接和纯化循环或链结构在长度和分支位置方面缺乏模块化的限制。在这里,我们建立了结构定义的异型多聚泛素链的一锅光控合成。我们设计了在用于聚合的赖氨酸残基处具有光不稳定保护基的泛素衍生物。受保护泛素单元的特定连锁酶促延伸和光诱导脱保护的重复循环使得能够逐步添加具有适当功能的泛素以控制长度和分支位置。分支的位置控制是在不分离中间体的情况下实现的,允许一锅法合成 K63 三泛素链和具有确定分支位置的 K63/K48 异型四泛素链。
更新日期:2023-05-18
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