The structure of an ancient genotype-phenotype map shaped the functional evolution of a protein family

Mutation is more likely to produce some phenotypes than others, but the causal role of these production propensities in the evolution of phenotypic diversity remains unclear. There are two major challenges: it is difficult to separate the effect of the genotype-phenotype (GP) map from that of natural selection when analyzing natural diversity, and most extant phenotypes evolved long ago in species whose GP maps cannot be recovered. Using reconstructed ancestral transcription factors, we created libraries containing all possible amino acid combinations at historically variable sites in the proteins’ DNA binding interface (the genotypes) and measured their capacity to specifically bind DNA elements containing all possible combinations of nucleotides at historically variable sites (the phenotypes). The two ancestral GP maps were strongly anisotropic (the distribution of phenotypes encoded by genotypes is highly nonuniform) and heterogeneous (the phenotypes accessible around each genotype v..., , , # Data from: The structure of an ancient genotype-phenotype map shaped the functional evolution of a protein family

[Access this dataset on Dryad](https://doi.org/10.5061/dryad.18931zd7m)

This dataset contains the raw read counts and the estimated mean fluorescence for all protein genotypes across all 16 response elements for three experimental replicates.

We used fluorescence-activated cell sorting (FACS) to separate cells based on their GFP expression. We performed two rounds of sorting: an initial “enrichment sort” to enrich for GFP+ variants in the full libraries, and a second, higher resolution “binned sort” on the enriched libraries to generate quantitative fluorescence estimates for each variant. To normalize fluorescence to cell volume, GFP gates were drawn to have a slope of 1.5 on a log(FSC-A)-log(GFP) plot. We sorted 2.5×10^7 cells per library in the enrichment stage.

The binned sort was performed to yield three replicates per library. Binned sorting followed the enrichme...,