High-resolution mapping of protein sequence-function relationships

Abstract

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 possessed 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.

A framework for exhaustively mapping functional missense variants

“…In Stage 4, like some previous DMS efforts (Doud & Bloom, 2016), we directly sequenced the coding region from the clone population to determine variant frequency before and after selection. Use of tiled amplicons enables individual template molecules to be sequenced on both strands, allowing elimination of most base‐calling errors (Fowler et al , 2010; Whitehead et al , 2012; Zhang et al , 2016) (see Materials and Methods for details). This reduction in base‐calling error allows us to more accurately measure lower allele frequencies in mutagenized libraries.…”

“…Deep mutational scanning (DMS) (Fowler et al , 2010; Fowler & Fields, 2014; Starita et al , 2014), a strategy for large‐scale functional testing of variants, can functionally annotate a large fraction of amino acid substitutions for a substantial subset of residue positions. Recent DMS studies, for example, covered the critical RING domain of BRCA1 (Starita et al , 2015) associated with breast cancer risk, and the PPARG protein associated with Mendelian lipodystrophy and increased risk of type 2 diabetes (Majithia et al , 2016).…”

Navigating cancer network attractors for tumor-specific therapy

“…Here we extend this definition to include a more fine-grained and precise definition of protein function as an ensemble of protein features that together describe the different functional capabilities of proteins (e.g., ATP binding, substrate specificity, protein activation or phospho-tyrosine binding). This new definition would not only adapt well to current studies of sequencefunction associations 15,16 , but also lead to a better description of the effects of a mutation affecting such residues (Fig. 2a,b).…”

Deep sequencing analysis of phage libraries using Illumina platform

“…For example, Fisher and co-workers recently demonstrated the use of Illumina sequencing to characterize phage-displayed libraries of single chain antibodies (scFv) [12]. Fields and co-worker used Illumina sequencing to characterize selection from libraries of WW-protein displayed on T7 phage [13]. Johan den Dunnen and co-worker used Illumina to characterize peptide libraries after one round of panning against cell surface receptors [14].…”