Large-scale mapping of the protein function landscape

描述（由申请人提供）：了解突变如何影响蛋白质功能对于理解天然蛋白质如何进化、工程化具有有用特性的新蛋白质以及预测疾病相关突变非常重要。上位性是一个突变的效果取决于另一个突变的存在的现象。这些上位相互作用阻碍了我们预测突变表型结果的能力，并限制了进化可用的突变途径。实验室进化研究强调了蛋白质功能的多面性，以及竞争性生物物理特性（如酶活性和蛋白质稳定性）如何在残基之间产生上位相互作用。我假设大多数上位性是由于这些多效性机制而产生的，而不是残基之间的直接物理相互作用。人尿素循环酶精氨酸酶I（hArgI）将用于研究序列变化如何影响表达、稳定性和酶活性。该实验方法将利用并行DNA测序和超高通量筛选的最新进展，以前所未有的规模绘制蛋白质功能图谱。这些数据将用于探索生物物理特性的组合如何产生突变上位性，并定义功能蛋白质序列的空间。

DESCRIPTION (provided by applicant): Knowledge of how mutations affect protein function is important for understanding how natural proteins evolved, engineering new proteins with useful properties, and predicting disease-associated mutations. Epistasis is the phenomenon where the effect of one mutation depends on the presence of another mutation. These epistatic interactions stymie our ability to predict the phenotypic consequences of mutations, and can restrict the mutational pathways that are available to evolution. Laboratory evolution studies have highlighted the multifaceted nature of protein function and how competing biophysical properties, such as enzymatic activity and protein stability, can generate epistatic interactions between residues. I hypothesize that most epistasis arises as a result of these pleiotropic mechanisms, rather than direct physical interactions between residues. The human urea cycle enzyme Arginase I (hArgI) will be used to study how sequence changes affect expression, stability, and enzymatic activity. The experimental approach will leverage recent advances in parallel DNA sequencing and ultra-high- throughput screening to map the protein function landscape on an unprecedented scale. These data will be used to explore how combinations of biophysical properties generate mutational epistasis and define the space of functional protein sequences.