Solution structure and dynamics of polyubiquitin chains

本研究旨在研究真核生物中普遍存在的蛋白质降解信号--多泛素(PolyUb)链的溶液结构和动力学特征。泛素-蛋白酶体蛋白分解途径是短寿命蛋白周转的主要调节机制，它影响着多种重要的细胞事件。了解26S蛋白酶体如何识别PolyUb链是理解调控机制的核心。尽管有重要证据表明，不同的PolyUb链的特异性识别是基于构象决定因素，但到目前为止，对于这些分子在溶液中的构象性质还没有明确的看法。获得这样的信息是绝对必要的，以便发展对不同链如何能够作为特定信号的分子理解。我们将使用核磁共振方法来确定四联体链的三维构象，该链在溶液中是自由的，并与蛋白酶体的模拟多肽结合。这将使用由自旋弛豫和剩余偶极耦合测量得出的长程、取向约束来执行。这些新的核磁共振方法将与基于NOES的传统方法结合使用，并基于化学位移扰动和溶剂可及性测量绘制结构域间或分子间界面。对于这些研究，我们将使用由细菌表达的Ub单体(未标记和统一标记)组装的分段同位素标记链，使用体外连接反应。我们将测量双Ub和四Ub中的蛋白质动力学，并与单体Ub进行比较，以确定结构域间运动的贡献，并表征多Ub链的构象灵活性。

This proposal is to characterize the solution structure and dynamics of polyubiquitin (polyUb) chains, a universal proteolytic signal in eukaryotes. The ubiquitin-proteasome proteolytic pathway is the principal regulatory mechanism for the turnover of short-lived proteins that influences a variety of vital cellular events. Understanding how polyUb chains are recognized by the 26S proteasome is central to understanding of the mechanisms of regulation. Although significant evidence suggests that the specific recognition of different polyUb chains is based on conformational determinants, as yet there is no clear view of the conformational properties of these molecules in solution. Obtaining such information is absolutely necessary in order to develop a molecular understanding of how different chains are able to act as specific signals. We will use NMR approaches to determine the three-dimensional conformation of the tetra-Ub chain free in solution and bound to a peptide mimic of the proteasome. This will be performed using long-range, orientational constraints derived from spin-relaxation and residual dipolar couplings measurements. These novel NMR approaches will be used in combination with the conventional methods based on NOES and mapping of the interdomain or intermolecular interface based on chemical shift perturbations and solvent accessibility measurements. For these studies, we will use segmentally isotope-labeled chains assembled from bacterially-expressed Ub monomers (unlabeled and uniformly labeled) using in vitro conjugation reaction. We will measure protein dynamics in the dual Ub and tetra-Ub, in comparison with that of the monomeric Ub, in order to determine the contribution from interdomain motions and characterize the conformational flexibility of polyUb chains.