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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.

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Figure 1: A highly parallel assay for exploring protein sequence-function relationships.
Figure 2: Comparison of mutational tolerance and evolutionary conservation in the WW domain.
Figure 3: Comprehensive sequence-function map of the WW domain.
Figure 4: Prediction of WW domain folding energies and double-mutant enrichment ratios.

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We thank C. Lee and J. Shendure for assistance with DNA sequencing, and J. Kelly, J. Thomas, J. Hesselberth, E. Phizicky, A. Rubin, L. Starita and K. McGarvey for helpful comments and discussion. This work was supported by the US National Institutes of Health (P41 RR11823 to S.F. and D.B., and F32GM084699 to D.M.F.). S.F. and D.B. were supported by the Howard Hughes Medical Institute.

Author information




D.M.F. conceived of the method, carried out the experiments, analyzed the data and wrote the paper; C.L.A. conceived of the method, analyzed the data and wrote the paper; J.J.S. carried out the experiments; E.H.K., S.J.F. and D.B. carried out the protein folding and binding energy calculations; and S.F. conceived of the method and wrote the paper.

Corresponding author

Correspondence to Stanley Fields.

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The authors declare no competing financial interests.

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Supplementary Text and Figures

Supplementary Figures 1–11, Supplementary Tables 1–2, Supplementary Note 1 (PDF 13072 kb)

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Fowler, D., Araya, C., Fleishman, S. et al. High-resolution mapping of protein sequence-function relationships. Nat Methods 7, 741–746 (2010).

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