HIGH-RESOLUTION MAPPING OF PROTEIN SEQUENCE-FUNCTION RELATIONSHIPS

蛋白质序列-功能关系的高分辨率绘图

• 批准号: 8365920
• 负责人: STANLEY FIELDS
• 金额: $ 2.18万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8365920
• 关键词: Amino Acid Sequence; Binding; Biological Assay; Biology; DNA Sequence; Funding; Fungal Genome; Grant; Human; In Vitro; Ligands; Maps; Mutation; National Center for Research Resources; Peptide Sequence Determination; Peptides; Performance; Phage Display; Positioning Attribute; Principal Investigator; Property; Proteins; Research; Research Infrastructure; Resolution; Resources; Source; United States National Institutes of Health; Variant; cost; in vivo; preference; protein function

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.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 我们提出了一种大规模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、适度选择活性以及高通量 DNA 测序来量化每个变体的性能。使用这种策略，我们通过噬菌体展示进行三轮和六轮选择后跟踪人类 WW 结构域的超过 600,000 个变体与其肽配体的结合性能。这些变体的结合特性定义了整个 WW 域突变偏好的高分辨率图谱；每个位置都有独特的特征，无法通过一些代表性的突变来捕获。我们的方法可应用于许多体外或体内蛋白质测定，为理解蛋白质功能与序列的关系提供了通用方法。