Solution structure and dynamics of polyubiquitin chains

多聚泛素链的溶液结构和动力学

• 批准号: 6622823
• 负责人: DAVID FUSHMAN
• 金额: $ 22.03万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6622823
• 关键词: Escherichia coli; active sites; binding sites; calorimetry; conformation; fluorimetry; mathematical model; nuclear magnetic resonance spectroscopy; peptide chemical synthesis; posttranslational modifications; proteasome; protein structure function; site directed mutagenesis; stable isotope; stable isotope double label; statistics /biometry; ubiquitin

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.

该建议是为了表征多泛素链的溶液结构和动力学，这是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短寿命蛋白营业的主要调节机制，影响了各种重要的细胞事件。 了解26S蛋白酶体如何识别多元链对于理解调节机制至关重要。 尽管大量证据表明，对不同的polyub链的特定识别是基于构象决定因素，但目前尚无清楚地看出这些分子在溶液中的构象性能。 获得此类信息是绝对必要的，以发展分子了解不同链如何充当特定信号的理解。 我们将使用NMR方法来确定溶液中四链链的三维构象，并与蛋白酶体的肽模仿结合。 这将使用从自旋 - 浮肿和残留偶极耦合测量的远程定向约束来进行。 这些新型的NMR方法将与基于NOE的常规方法以及基于化学位移扰动和溶剂可访问性测量的常规方法结合使用。 在这些研究中，我们将使用从细菌表达的UB单体（未标记和均匀标记）组装的分段同位素标记的链中使用体外共轭反应。 与单体UB相比，我们将测量双UB和TETRA-UB中的蛋白质动力学，以确定域间运动的贡献并表征Polyub链的构象灵活性。