Solution structure and dynamics of polyubiquitin chains

多聚泛素链的溶液结构和动力学

• 批准号: 7937184
• 负责人: DAVID FUSHMAN
• 金额: $ 23.26万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7937184
• 关键词: 26S proteasome; Address; Amaze; Binding; Cell Cycle; Cells; Chemicals; Complement; Complex; Coupling; Epitopes; Eukaryota; Event; Immunity; Inflammation; Label; Life; Ligand Binding; Link; Lysine; Malignant - descriptor; Maps; Measurement; Measures; Methods; Molecular; Molecular Conformation; Motion; Mutation; Pathway interactions; Peptides; Play; Polyubiquitin; Positioning Attribute; Process; Property; Protein Dynamics; Proteins; Regulation; Relative (related person); Relaxation; Research Personnel; Residual state; Role; Signal Transduction; Solutions; Solvents; Specificity; Structure; Structure-Activity Relationship; Temperature; UBA Domain; Ubiquitin; Work; base; design; flexibility; inhibitor/antagonist; method development; monomer; multicatalytic endopeptidase complex; novel; programs; receptor; response; three dimensional structure

DESCRIPTION (provided by applicant): This proposal is to characterize the solution structure, dynamics, and interactions 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 Lys48-linked polyUb chains are recognized by the 26S proteasome and other downstream effector molecules is central to our understanding of the mechanisms of regulation. Remarkably, polyUb chains linked via different lysines also act as cellular signals, although regulatory rather than proteolytic. 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 Lys48-linked tetra-Ub chain, free in solution, and of di- and tetra-Ub bound to molecular mimics of the proteasomal receptors, hHR23A and S5a. This will be performed using a combination of long-range, orientational constraints derived from spin-relaxation and residual dipolar coupling measurements, complemented with distance information from paramagnetic relaxation enhancements and pseudocontact shifts introduced by paramagnetic labels. 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. We will use a representative set of UBA domains in order to identify structural determinants of the linkage specificity of various Ub-chain receptors. Domain motions play essential role in polyUb chain's ability to interact with various receptors. We will introduce mutations in the residues at the Ub-Ub interface in order to identify determining factors contributing to the interface stability and dynamics. We will measure protein dynamics in di-Ub and tetra-Ub under various pH and temperature conditions in order to determine the contribution from interdomain motions and to characterize the conformational flexibility of polyUb chains.

描述（由申请人提供）：该建议是为了表征溶液结构，动力学和多泛素（polyub）链，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蛋白酶体和其他下游效应分子如何识别Lys48连接的多核链是我们对调节机制的理解至关重要的。值得注意的是，通过不同赖氨酸连接的多蛋白链也充当细胞信号，尽管调节而不是蛋白水解。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Lys48连接的四链​​链的三维构象，溶液中的自由，以及与蛋白酶体受体HHR23A和S5A的分子模拟物结合的DI-和TETRA-UB。这将使用源自自旋 - 浮肿和残余偶极耦合测量的远程，定向约束的组合进行，并与顺磁性标签引入的顺磁性松弛增强和伪内膜移动相辅相成。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。我们将使用一组代表性的UBA域，以确定各种UB链受体的连锁特异性的结构决定因素。域运动在Polyub链与各种受体相互作用的能力中起着至关重要的作用。我们将在UB-UB界面的残基中引入突变，以确定有助于界面稳定性和动力学的决定因素。我们将在各种pH和温度条件下测量DI-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链的构象柔韧性。