The Impact of Mutation on the Conformations and Recognition of Ubiquitin

突变对泛素构象和识别的影响

• 批准号: 8728042
• 负责人: James Solomon Fraser
• 金额: $ 38.63万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-20 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8728042
• 关键词: Active Sites; Address; Affect; Binding; Biophysics; Catalysis; Cells; Complement; Coupled; Custom; Data; Data Collection; Defect; Disease; Enzymes; Eukaryota; Evaluation; Future; Genetic; Hereditary Disease; In Vitro; Individual; Knowledge; Libraries; Lysine; Maps; Measures; Mediating; Methods; Modeling; Molecular; Molecular Conformation; Monitor; Mutate; Mutation; Peptidylprolyl Isomerase; Phenotype; Polyubiquitin; Population; Protein Conformation; Protein Engineering; Proteins; Relative (related person); Resolution; Roentgen Rays; Role; Saccharomyces cerevisiae; Sampling; Side; Site; Specificity; Speed; Structure; Surface; Temperature; Testing; Ubiquitin; Work; X-Ray Crystallography; Yeasts; abstracting; base; biophysical model; cost; design; disease-causing mutation; drug discovery; electron density; engineering design; genome sequencing; improved; in vivo; interest; molecular recognition; mutant; protein function; protein protein interaction; protein structure; research study; response; small molecule; tool

Project Summary/Abstract Understanding how mutations affect protein structure significantly impacts drug discovery, protein engineering, and the interpretation of individual genome sequences. However, the effects of many mutations, whether they are beneficial or deleterious, cannot be understood from static protein structures alone. This problem is especially significant for mutations that are located far away from active sites and interaction surfaces. If these mutations do not have obvious large stability costs and are remote from functional sites, how can they influence protein function? Rather than affecting the average structure defined by traditional X-ray crystallography, this proposal determines how mutations may change the relative population of alternative conformations. However, identifying alternative conformations and measuring their impact on protein function represents an experimental challenge. To address these problems, this project builds on my methodological advances to reveal alternative conformations by room temperature X-ray crystallography and electron density sampling. I will study the protein-protein interactions of ubiquitin (Ub) in S. cerevisiae as a model to understand how perturbing the relative populations of conformations impacts molecular recognition. Ub is an ideal model to study the importance of alternative conformations because: previous studies have indicated that diverse Ub conformations and poly-Ub linkages mediate distinct functional roles; its remarkable sequence and functional conservation suggests that the populations of alternative conformations will be particularly susceptible to mutation; and it is a small protein that can be comprehensively mutated. Moreover, I have generated preliminary high-resolution room temperature X-ray data that complement previous NMR experiments to define its accessible alternative conformations. Despite the central importance of different Ub conformations for the cell, the general principles of how different Ub conformations are recognized and direct the assembly of poly-Ub chains remain to be elucidated. To determine how mutations can affect the assembly of specific poly-Ub chains, I will monitor how alternative side chain conformations of Ub participate in the catalytic mechanism of the E2 Ubc1. To test how mutations afect Ub interactions in vivo, I will measure a unique phenotypic profile for each Ub mutant. The central role of Ub in proteostasis and its sequence conservation suggest that the principles I uncover will be widely applicable across all eukaryotes. By measuring the impact of mutation on the conformational ensemble, this proposal addresses fundamental biophysical models of interaction specificity, the organization of the Ub-interaction network, and the molecular mechanisms of phenotypic change. Predictions of how mutation can change the relative populations of conformations are especially important as increased sequencing efforts provide the genetic basis for rare genetic diseases. This project will improve our knowledge and understanding of the relationship between mutation, alternative conformations, and phenotype.

项目总结/摘要 了解突变如何影响蛋白质结构对药物发现，蛋白质工程， 以及对个体基因组序列的解读。然而，许多突变的影响，无论它们是否 是有益的还是有害的，不能仅仅从静态的蛋白质结构来理解。这个问题 对于远离活性位点和相互作用表面的突变尤其重要。如果这些 突变没有明显的大的稳定性成本，并且远离功能位点，它们如何能够 影响蛋白质功能？而不是影响传统X射线定义的平均结构 晶体学，这一建议确定如何突变可能会改变相对人口的替代 构象然而，识别替代构象并测量其对蛋白质功能的影响， 是一个实验性的挑战。为了解决这些问题，这个项目建立在我的方法论基础上， 用室温X射线晶体学和电子密度揭示交替构象的进展 取样.本论文主要研究S.酿酒酵母作为一个模型， 了解如何扰乱构象的相对群体影响分子识别。UB是一个 理想的模型来研究替代构象的重要性，因为：以前的研究表明， 不同的Ub构象和poly-Ub键介导不同的功能作用;其显著的序列和 功能保守性表明，替代构象的种群将特别 易突变，是一种可以全面突变的小蛋白。而且我 生成了初步的高分辨率室温X射线数据，补充了以前的NMR 实验来确定其可接近的替代构象。尽管不同的UB的核心重要性 不同的Ub构象如何被识别和指导的一般原则， 聚-Ub链的组装仍有待阐明。以确定突变如何影响程序集 具体的poly-Ub链，我将监测如何替代侧链构象的Ub参与 E2 Ubc 1的催化机制。为了测试突变如何影响Ub在体内的相互作用，我将测量 每个Ub突变体都有独特的表型。Ub在蛋白质稳态中的中心作用及其序列 保护表明，我发现的原则将广泛适用于所有真核生物。通过 测量突变对构象系综的影响，该建议解决了基本的 相互作用特异性的生物物理模型，Ub相互作用网络的组织，以及分子生物学模型。 表型变化的机制。预测突变如何改变 构象是特别重要的，因为增加测序工作提供了罕见的遗传基础。 遗传病这个项目将提高我们的知识和理解之间的关系， 突变、替代构象和表型。