Local fitness landscape of the green fluorescent protein

Comprehensive genotype–phenotype mapping of the green fluorescent protein shows that the local fitness peak is narrow, shaped by a high prevalence of epistatic interactions, providing for the loss of fluorescence when the joint effect of mutations exceeds a threshold. Fyodor Kondrashov and colleagues report comprehensive genotype–phenotype mapping across an entire protein, based on analysis of the fitness landscape of green fluorescent protein (GFP) using a molecular barcoding and sequencing approach. They find that the fitness landscape is characterized by locally narrow regions, combined with a high prevalence of epistatic interactions, providing for the loss of fluorescence when the joint effect of mutations exceeds a threshold. Fitness landscapes1,2 depict how genotypes manifest at the phenotypic level and form the basis of our understanding of many areas of biology2,3,4,5,6,7, yet their properties remain elusive. Previous studies have analysed specific genes, often using their function as a proxy for fitness2,4, experimentally assessing the effect on function of single mutations and their combinations in a specific sequence2,5,8,9,10,11,12,13,14,15 or in different sequences2,3,5,16,17,18. However, systematic high-throughput studies of the local fitness landscape of an entire protein have not yet been reported. Here we visualize an extensive region of the local fitness landscape of the green fluorescent protein from Aequorea victoria (avGFP) by measuring the native function (fluorescence) of tens of thousands of derivative genotypes of avGFP. We show that the fitness landscape of avGFP is narrow, with 3/4 of the derivatives with a single mutation showing reduced fluorescence and half of the derivatives with four mutations being completely non-fluorescent. The narrowness is enhanced by epistasis, which was detected in up to 30% of genotypes with multiple mutations and mostly occurred through the cumulative effect of slightly deleterious mutations causing a threshold-like decrease in protein stability and a concomitant loss of fluorescence. A model of orthologous sequence divergence spanning hundreds of millions of years predicted the extent of epistasis in our data, indicating congruence between the fitness landscape properties at the local and global scales. The characterization of the local fitness landscape of avGFP has important implications for several fields including molecular evolution, population genetics and protein design.