High resolution mapping of protein sequence-function relationships in Human Yap65 CA0002

We present a large-scale approach to investigate the functional consequences of sequence variation in a protein. The approach entails the display of hundreds of thousands of protein variants, moderate selection for activity, and high throughput DNA sequencing to quantify the performance of each variant. Using this strategy, we tracked the performance of >600,000 variants of a human WW domain after three and six rounds of selection by phage display for binding to its peptide ligand. Binding properties of these variants defined a high-resolution map of mutational preference across the WW domain; each position possessed unique features that could not be captured by a few representative mutations. Our approach could be applied to many in vitro or in vivo protein assays, providing a general means for understanding how protein function relates to sequence.

本研究提出一种大规模研究方法，用于探究蛋白质序列变异所带来的功能影响。该方法涵盖数十万种蛋白质变体的展示、基于活性的适度筛选，以及用于量化各变体性能的高通量DNA测序技术。借助该策略，我们针对人类WW结构域（WW domain）的超过60万个变体，在通过噬菌体展示（phage display）进行3轮和6轮筛选以结合其肽配体（peptide ligand）后，追踪了各变体的性能表现。这些变体的结合特性，构建出了WW结构域全域突变偏好的高分辨率图谱；每个位点均具备独特特征，无法通过少量代表性突变加以体现。本研究方法可应用于众多体外（in vitro）或体内（in vivo）蛋白质实验，为解析蛋白质功能与序列之间的关联提供了通用途径。