The Impact of Mutation on the Conformations and Recognition of Ubiquitin

描述（由申请人提供）：了解突变如何影响蛋白质结构会显著影响药物发现、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，无论是有益的还是有害的，都不能单独从静态蛋白质结构中理解。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，它们如何影响蛋白质功能？这个建议不是影响传统X射线晶体学定义的平均结构，而是确定突变如何改变替代构象的相对数量。然而，确定替代构象和测量它们对蛋白质功能的影响是一个实验挑战。为了解决这些问题，这个项目建立在我的方法进步，揭示替代构象的室温X射线晶体学和电子密度采样。本论文主要研究S.酿酒酵母作为一个模型，以了解如何扰乱构象的相对群体影响分子识别。Ub是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poly-Ub键介导不同的功能作用;其显着的序列和功能保守性表明，替代构象的群体将特别容易发生突变;它是一个小的蛋白质，可以全面突变。此外，我已经产生了初步的高分辨率室温X射线数据，补充以前的NMR实验，以确定其可访问的替代构象。尽管不同的Ub构象的细胞的核心重要性，不同的Ub构象如何识别和指导多聚Ub链的组装的一般原则仍有待阐明。为了确定突变如何影响特定聚Ub链的组装，我将监测Ub的替代侧链构象如何参与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突变体的独特表型。Ub在蛋白质稳定中的核心作用及其序列保守性表明，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和表型变化的分子机制。预测突变如何改变构象的相对群体尤其重要，因为测序工作的增加为罕见遗传疾病提供了遗传基础。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。 公共卫生相关性：该提案描述了测量和预测突变引起的蛋白质构象变化的新方法。蛋白质构象如何被致病突变扰乱的知识，加上恢复正确蛋白质构象的方法，将大大增加治疗疾病的机会。

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.