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，对药物发现、蛋白质工程和个体基因组序列的解释产生重大影响。然而，许多突变的影响，无论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，并且远离功能位点，它们如何影响蛋白质的功能？这一建议不是影响传统X射线结晶学定义的平均结构，而是确定突变可能如何改变替代构象的相对总体。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，这个项目建立在我的方法学进步的基础上，通过室温X射线结晶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，以了解扰乱相对构象群体如何影响分子识别。Ub是研究替代构象重要性的理想模型，因为：以前的研究表明，多样化的Ub构象和多Ub连接介导了不同的功能角色；其显著的序列和功能保守表明替代构象的种群将特别容易发生突变；并且它是一种可以全面突变的小蛋白。此外，我已经生成了初步的高分辨率室温X射线数据，这些数据补充了之前的核磁共振实验，以确定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构象并指导多聚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的PolyUb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体内的Ub相互作用，我将测量每个Ub突变体的独特表型特征。Ub在蛋白平衡及其序列保守中的核心作用表明，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整体的影响，这一建议解决了相互作用专一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。预测突变如何改变构象的相对种群尤其重要，因为更多的测序工作为罕见的遗传病提供了遗传基础。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。 与公共卫生相关：这项建议描述了测量和预测突变引起的蛋白质构象变化的新方法。了解致病突变是如何扰乱蛋白质构象的，再加上恢复正确蛋白质构象的方法，将极大地扩大治疗疾病的机会。

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.