Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, University of Maryland, College Park, Maryland 20742, United States.
J Am Chem Soc. 2011 Nov 9;133(44):17855-68. doi: 10.1021/ja207220g. Epub 2011 Oct 19.
Polymeric chains made of a small protein ubiquitin act as molecular signals regulating a variety of cellular processes controlling essentially all aspects of eukaryotic biology. Uncovering the mechanisms that allow differently linked polyubiquitin chains to serve as distinct molecular signals requires the ability to make these chains with the native connectivity, defined length, linkage composition, and in sufficient quantities. This, however, has been a major impediment in the ubiquitin field. Here, we present a robust, efficient, and widely accessible method for controlled iterative nonenzymatic assembly of polyubiquitin chains using recombinant ubiquitin monomers as the primary building blocks. This method uses silver-mediated condensation reaction between the C-terminal thioester of one ubiquitin and the ε-amine of a specific lysine on the other ubiquitin. We augment the nonenzymatic approaches developed recently by using removable orthogonal amine-protecting groups, Alloc and Boc. The use of bacterially expressed ubiquitins allows cost-effective isotopic enrichment of any individual monomer in the chain. We demonstrate that our method yields completely natural polyubiquitin chains (free of mutations and linked through native isopeptide bonds) of essentially any desired length, linkage composition, and isotopic labeling scheme, and in milligram quantities. Specifically, we successfully made Lys11-linked di-, tri-, and tetra-ubiquitins, Lys33-linked diubiquitin, and a mixed-linkage Lys33,Lys11-linked triubiquitin. We also demonstrate the ability to obtain, by high-resolution NMR, residue-specific information on ubiquitin units at any desired position in such chains. This method opens up essentially endless possibilities for rigorous structural and functional studies of polyubiquitin signals.
由小蛋白泛素组成的聚合链作为分子信号，调节控制真核生物生物学各个方面的各种细胞过程。揭示允许不同连接的多泛素链作为不同的分子信号的机制，需要能够以天然连接、定义的长度、连接组成和足够的数量来制造这些链。然而，这一直是泛素领域的一个主要障碍。在这里，我们提出了一种稳健、高效、广泛适用的方法，用于使用重组泛素单体作为主要构建块，进行可控的迭代非酶聚合多泛素链。该方法使用银介导的 C 末端硫酯和另一个泛素上特定赖氨酸的 ε-氨基之间的缩合反应。我们通过使用可去除的正交胺保护基 Alloc 和 Boc 来增强最近开发的非酶方法。使用细菌表达的泛素可以使链中任何单个单体的同位素富集具有成本效益。我们证明，我们的方法可以得到完全天然的多泛素链（无突变且通过天然异肽键连接），其长度、连接组成和同位素标记方案基本上是任意的，并且可以毫克级的量获得。具体来说，我们成功地制备了 Lys11 连接的二、三、四泛素，Lys33 连接的二泛素和混合 Lys33，Lys11 连接的三泛素。我们还证明了通过高分辨率 NMR 获得此类链中任何所需位置的泛素单位的残基特异性信息的能力。该方法为严格的多泛素信号的结构和功能研究开辟了无限可能。
