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.
Submitter: Connie Wang
Submission Date: Oct. 24, 2016, 4:27 p.m.
|Number of data points||416|
|Proteins||WW domain hYAP65|
|Assays/Quantities/Protocols||Experimental Assay: Phage display binding and then sequencing abundance|
|Libraries||Allowed mutations in 25 residue region due to illumina restraints, only looking at single mutants here|