Solution structure and dynamics of polyubiquitin chains

这个建议是表征的解决方案的结构和动力学的聚泛素（polyUb）链，在真核生物中的通用蛋白水解信号。 泛素-蛋白酶体蛋白水解途径是影响各种重要细胞事件的短寿命蛋白质周转的主要调节机制。 了解polyUb链如何被26 S蛋白酶体识别对于理解调控机制至关重要。 虽然有显著的证据表明，不同的polyUb链的特异性识别是基于构象决定因素，但迄今为止，这些分子在溶液中的构象特性还没有明确的观点。 获得这些信息是绝对必要的，以发展一个分子的理解，不同的链是如何能够作为特定的信号。 我们将使用NMR方法来确定在溶液中游离的四-Ub链的三维构象，并与蛋白酶体的肽模拟物结合。 这将使用来自自旋弛豫和残余偶极耦合测量的长范围，取向约束。 这些新的NMR方法将与基于NOES的常规方法和基于化学位移扰动和溶剂可及性测量的域间或分子间界面的映射结合使用。 对于这些研究，我们将使用体外偶联反应，使用由细菌表达的Ub单体（未标记和均匀标记）组装的节段同位素标记链。 我们将测量蛋白质动力学的双Ub和四Ub，在与单体Ub相比，以确定从域间运动的贡献和表征polyUb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.