HIGH-RESOLUTION MAPPING OF PROTEIN SEQUENCE-FUNCTION RELATIONSHIPS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 我们提出了一个大规模的方法来研究蛋白质序列变异的功能后果。该方法需要展示数十万种蛋白质变体，适度选择活性和高通量DNA测序以量化每个变体的性能。使用这种策略，我们在通过噬菌体展示进行三轮和六轮选择后追踪了> 600，000个人WW结构域变体与其肽配体结合的性能。这些变体的结合特性定义了整个WW结构域的突变偏好的高分辨率图;每个位置具有无法被少数代表性突变捕获的独特特征。我们的方法可以应用于许多体外或体内蛋白质测定，提供了一个一般的手段，了解蛋白质功能如何与序列相关。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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 had 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.