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.

项目摘要/摘要 了解突变如何影响蛋白质结构会显着影响药物发现，蛋白质工程， 以及单个基因组序列的解释。但是，许多突变的影响，是否 是有益的或有害的，不能仅凭静态蛋白质结构来理解。这个问题是 对于远离活性位点和相互作用表面的突变特别重要。如果这些 突变没有明显的稳定成本明显，并且远离功能站点，它们如何才能 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，该提案决定了突变如何改变替代的相对种群 构象。但是，确定替代构象并测量其对蛋白质功能的影响 代表一个实验挑战。为了解决这些问题，该项目建立在我的方法论基础上 通过室温X射线晶体学和电子密度揭示替代构象的进步 采样。我将研究酿酒酵母中泛素（UB）的蛋白质 - 蛋白质相互作用作为模型 了解构象相对群体的扰动如何影响分子识别。 ub是一个 研究替代构象重要性的理想模型，因为：以前的研究表明 不同的UB构象和多核链接介导了不同的功能角色；它的显着序列和 功能保护表明，替代构象的种群尤其是 容易受到突变；它是一种可以全面突变的小蛋白质。而且，我有 生成的初步高分辨率室温X射线数据，与先前的NMR相辅相成 定义其可访问的替代构象的实验。尽管不同的UB具有至关重要的重要性 细胞的构象，这是如何识别不同UB构象和直接的一般原理 多核链的组装仍有待阐明。确定突变如何影响组装 在特定的多核链中，我将监视UB的替代侧链构象如何参与 E2 UBC1的催化机制。为了测试突变如何与体内的UB相互作用相比，我将测量 每个UB突变体的独特表型曲线。 UB在蛋白抑制及其序列中的核心作用 保护表明，我发现的原则将在所有真核生物中广泛适用。经过 该提案衡量突变对构象合奏的影响，解决了基本 相互作用特异性的生物物理模型，UB互动网络的组织和分子 表型变化的机制。突变如何改变相对种群的预测 构象特别重要，因为增加的测序工作为罕见的遗传基础提供了 遗传疾病。该项目将提高我们对之间关系的知识和理解 突变，替代构象和表型。