High-resolution mapping of protein sequence-function relationships.
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.
Fowler
Douglas M
DM
Department of Genome Sciences, University of Washington, Seattle, WA, USA.
Araya
Carlos L
CL
Fleishman
Sarel J
SJ
Kellogg
Elizabeth H
EH
Stephany
Jason J
JJ
Baker
David
D
Fields
Stanley
S
D004247:DNA / Q000235:genetics; D030561:Databases, Nucleic Acid; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D019151:Peptide Library; D040081:Protein Array Analysis / Q000379:methods*; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013329:Structure-Activity Relationship
D019151:Peptide Library; D011506:Proteins; D004247:DNA