The Impact of Mutation on the Conformations and Recognition of Ubiquitin

项目摘要/摘要 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、 以及个体基因组序列的解释。然而，许多突变的影响，无论是它们 是有益的还是有害的，不能仅从静态的蛋白质结构来理解。这个问题是 对于远离活性部位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，而且远离功能位点，它们怎么可能 会影响蛋白质功能吗？而不是影响传统X射线定义的平均结构 结晶学，这一建议决定了突变可能如何改变替代的相对种群 构象。然而，确定替代构象并测量它们对蛋白质功能的影响 代表着一项实验性的挑战。为了解决这些问题，这个项目建立在我的方法论 室温X射线结晶学和电子密度法揭示交替构象的研究进展 取样。我将研究酿酒酵母中泛素(Ub)的蛋白质-蛋白质相互作用，作为一种模型 了解扰乱构象的相对群体如何影响分子识别。UB是一个 研究替代构象重要性的理想模型，因为：先前的研究表明 不同的Ub构象和多Ub连接调节不同的功能作用；其显着的序列和 功能守恒表明，替代构象的种群将特别 很容易发生突变；而且它是一种可以全面突变的小蛋白质。而且，我有 生成了补充先前核磁共振的初步高分辨率室温X射线数据 确定其可接近的替代构象的实验。尽管不同的Ub具有核心重要性 细胞的构象，如何识别和指导不同的Ub构象的一般原则 多聚-Ub链的组装还有待阐明。以确定突变如何影响组装 在特定的多Ub链中，我将监测Ub的替代侧链构象如何参与 E2Ubc1的催化机制。为了测试突变如何影响体内的Ub相互作用，我将测量一个 每个Ub突变体的唯一表型特征。Ub在蛋白平衡中的核心作用及其序列 保守主义表明，我发现的原理将广泛适用于所有真核生物。通过 衡量突变对构象集合的影响，这一建议解决了基本问题 相互作用专一性的生物物理模型，Ub相互作用网络的组织，以及分子 表型变化的机制。关于突变如何改变人类相对种群的预测 构象尤其重要，因为增加的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对两国关系的认识和理解 突变、交替构象和表型。

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.