Count selected

Binding

Count/Number

unitless

Illumina Sequencing

None

all possible combinations at 4 pos in Protein GB1: 39,40,41,54 were studied (these sites are in epistatic region as determined from previous pairwise study.

The “fitness” of protein GB1 variants, as determined by both stability (i.e. the fraction of folded proteins) and function (i.e. binding affinity to IgG-Fc), was measured in a high-throughput manner by coupling mRNA display (peptides are covalently linked to the encoding mRNAs) with Illumina deep sequencing.

The relative frequency of mutant sequences before and after selection allowed us to compute the fitness of each variant relative to the wild type protein (WT).