Department of Chemistry, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin 53706-1322, USA.
J Am Chem Soc. 2012 Apr 25;134(16):6916-9. doi: 10.1021/ja300500a. Epub 2012 Apr 17.
Chemical methods for modifying proteins can enable studies aimed at uncovering biochemical function. Herein, we describe the use of thiol-ene coupling (TEC) chemistry to report on the function of branched (also referred to as forked) ubiquitin trimers. We show how site-specific isopeptide (Nε-Gly-L-homothiaLys) bonds are forged between two molecules of Ub, demonstrating the power of TEC in protein conjugation. Moreover, we demonstrate that the Nε-Gly-L-homothiaLys isopeptide bond is processed to a similar extent by deubiquitinases (DUBs) as that of a native Nε-Gly-L-Lys isopeptide bond, thereby establishing the utility of TEC in the generation of Ub-Ub linkages. TEC is then applied to the synthesis of branched Ub trimers. Interrogation of these branched derivatives with DUBs reveals that the relative orientation of the two Ub units has a dramatic impact on how they are hydrolyzed. In particular, cleavage of K48C-linkages is suppressed when the central Ub unit is also conjugated through K6C, whereas cleavage proceeds normally when the central unit is conjugated through either K11C or K63C. The results of this work presage a role for branched polymeric Ub chains in regulating linkage-selective interactions.
化学修饰蛋白质的方法可以用于研究揭示生物化学功能。在这里，我们描述了使用硫醇-烯（TEC）偶联化学来研究分支（也称为叉形）泛素三聚体的功能。我们展示了如何在两个 Ub 分子之间形成特定位置的异肽（Nε-Gly-L-homothiaLys）键，证明了 TEC 在蛋白质偶联中的强大功能。此外，我们还证明了 Nε-Gly-L-homothiaLys 异肽键被去泛素化酶（DUBs）切割的程度与天然 Nε-Gly-L-Lys 异肽键相似，从而确立了 TEC 在生成 Ub-Ub 连接中的实用性。然后，TEC 被应用于合成分支 Ub 三聚体。用 DUBs 对这些分支衍生物进行分析表明，两个 Ub 单元的相对取向对它们的水解方式有显著影响。特别是，当中心 Ub 单元通过 K6C 进行缀合时，K48C 键的切割被抑制，而当中心单元通过 K11C 或 K63C 进行缀合时，切割则正常进行。这项工作的结果预示着分支聚合泛素链在调节连接选择性相互作用中的作用。
