Differential strengths of molecular determinants guide environment specific mutational fates

Organisms maintain competitive fitness in the face of environmental challenges through molecular evolution. However, it remains largely unknown how different biophysical factors constrain molecular evolution in a given environment. Here, using deep mutational scanning, we quantified empirical fitness of >2000 single site mutants of the Gentamicin-resistant gene (GmR) in Escherichia coli, in a representative set of physical (non-native temperatures) and chemical (small molecule supplements) environments. From this, we could infer how different biophysical parameters of the mutations constrain molecular function in different environments. We find ligand binding, and protein stability to be the best predictors of mutants’ fitness, but their relative predictive power differs across environments. While protein folding emerges as the strongest predictor at minimal antibiotic concentration, ligand binding becomes a stronger predictor of mutant fitness at higher concentration. Remarkably, strengths of environment-specific selection pressures were largely predictable from the degree of mutational perturbation of protein folding and ligand binding. By identifying structural constraints that act as determinants of fitness, our study thus provides coarse mechanistic insights into the environment specific accessibility of mutational fates.