Conformational switching during the production of branched polyubiquitin chains (A13*)

泛素链的翻译后修饰是真核蛋白质调控的一种主要形式。尽管人们对连接泛素和靶蛋白的E1-E2-E3三酶级联了解很多，但对特定泛素链形成的动态结构机制--尤其是E4酶的作用--知之甚少。在这个项目中，我们将研究在通过E1-E2-E3-E4四酶级联产生和识别特定的“分支”泛素链(其中单个泛素分子的赖氨酸29和48都是与额外泛素分子键合的异肽)过程中的构象转换机制。生物化学、化学、X射线结晶学和低温EM将被用来阐明三个单独的泛素由E2-E3和E2-E4酶对定点特异性连接的动态构象循环。此外，还将确定多泛素化的原理。

Post-translational modification by ubiquitin chains is a major form of eukaryotic protein regulation. Although much is known about E1-E2-E3 trienzyme cascades linking ubiquitin to target proteins, the dynamic structural mechanisms underlying formation of specific ubiquitin chains – and particularly roles of E4 enzymes – are poorly understood. In this project, we will investigate the mechanisms of conformational switching during generation and recognition of specific "branched" ubiquitin chains (wherein lysines 29 and 48 of an individual molecule of ubiquitin are both isopeptide bonded to additional ubiquitin molecules) by E1-E2-E3-E4 tetraenzyme cascades. Biochemistry, chemistry, X-ray crystallography, and cryo EM will be used to elucidate the dynamic conformational cycles by which three individual ubiquitins are site-specifically adjoined by E2-E3 and E2-E4 enzyme pairs. Furthermore, principles of polyubiquitylation will be determined.