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.

Studies of protein fitness landscapes reveal biophysical constraints guiding protein evolution and empower prediction of functional proteins. However, generalisation of these findings is limited due to scarceness of systematic data on fitness landscapes of proteins with a defined evolutionary relationship. We characterized the fitness peaks of four orthologous fluorescent proteins with a broad range of sequence divergence. While two of the four studied fitness peaks were sharp, the other two were considerably flatter, being almost entirely free of epistatic interactions. Mutationally robust proteins, characterized by a flat fitness peak, were not optimal templates for machine-learning-driven protein design – instead, predictions were more accurate for fragile proteins with epistatic landscapes. Our work paves insights for practical application of fitness landscape heterogeneity in protein engineering.

A novel method of protein labeling uses the highly dynamic reversible association of a cell-permeable fluorogenic dye and lipocalin Blc mutants.

Genetically encoded photosensitizers, proteins that produce reactive oxygen species when illuminated with visible light, are increasingly used as optogenetic tools. Their applications range from ablation of specific cell populations to precise optical inactivation of cellular proteins. Here, we report an orange mutant of red fluorescent protein KillerRed that becomes toxic when illuminated with blue or green light. This new protein, KillerOrange, carries a tryptophan-based chromophore that is novel for photosensitizers. We show that KillerOrange can be used simultaneously and independently from KillerRed in both bacterial and mammalian cells offering chromatic orthogonality for light-activated toxicity.