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: 8213132
• 负责人: James Solomon Fraser
• 金额: $ 33.18万
• 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/8213132
• 关键词: Active Sites; Address; Affect; Binding; 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; base; 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

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: This proposal describes new methods for measuring and predicting changes in protein conformations caused by mutation. Knowledge of how protein conformations are perturbed by disease-causing mutations, coupled with methods for restoring proper protein conformations would dramatically expand opportunities to treat disease.

描述（由申请人提供）：了解突变如何影响蛋白质结构显著影响药物发现，蛋白质工程和个体基因组序列的解释。然而，许多突变的影响，无论它们是有益的还是有害的，都不能仅从静态蛋白质结构中理解。这个问题对于位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，它们是如何影响蛋白质功能的呢？而不是影响由传统x射线晶体学定义的平均结构，该建议确定突变如何改变替代构象的相对种群。然而，确定替代构象并测量它们对蛋白质功能的影响是一项实验挑战。为了解决这些问题，本项目建立在我的方法进步的基础上，通过室温x射线晶体学和电子密度采样来揭示替代构象。我将研究酿酒酵母中泛素（Ub）的蛋白质-蛋白质相互作用，作为一个模型，以了解干扰相对群体的构象如何影响分子识别。Ub是研究替代构象重要性的理想模型，因为：先前的研究表明，不同的Ub构象和多Ub键介导不同的功能作用；其显著的序列和功能守恒表明，不同构象的群体将特别容易发生突变；它是一种可以全面突变的小蛋白质。此外，我已经生成了初步的高分辨率室温x射线数据，补充了以前的核磁共振实验，以定义其可访问的替代构象。尽管不同的Ub构象对细胞至关重要，但如何识别不同的Ub构象并指导聚Ub链的组装的一般原理仍有待阐明。为了确定突变如何影响特定多聚Ub链的组装，我将监测Ub的替代侧链构象如何参与E2 Ubc1的催化机制。为了测试突变如何影响Ub在体内的相互作用，我将测量每个Ub突变体的独特表型。Ub在蛋白质静止及其序列保护中的核心作用表明，我发现的原理将广泛适用于所有真核生物。通过测量突变对构象集合的影响，本研究提出了相互作用特异性的基本生物物理模型、ub相互作用网络的组织以及表型变化的分子机制。随着越来越多的测序工作为罕见的遗传疾病提供了遗传基础，预测突变如何改变相对的构象群体尤为重要。这个项目将提高我们对突变、替代构象和表型之间关系的认识和理解。