Differential strengths of molecular determinants guide environment specific mutational fates

Under the influence of selection pressures imposed by natural environments, organisms maintain competitive fitness through underlying molecular evolution of individual genes across the genome. For molecular evolution, how multiple interdependent molecular constraints play a role in determination of fitness under different environmental conditions is largely unknown. Here, using Deep Mutational Scanning (DMS), we quantitated empirical fitness of ∼2000 single site mutants of Gentamicin-resistant gene (GmR). This enabled a systematic investigation of effects of different physical and chemical environments on the fitness landscape of the gene. Molecular constraints of the fitness landscapes seem to bear differential strengths in an environment dependent manner. Among them, conformity of the identified directionalities of the environmental selection pressures with known effects of the environments on protein folding proves that along with substrate binding, protein stability is the common strong constraint of the fitness landscape. Our study thus provides mechanistic insights into the molecular constraints that allow accessibility of mutational fates in environment dependent manner.Author SummaryEnvironmental conditions play a central role in both organismal adaptations and underlying molecular evolution. Understanding of environmental effects on evolution of genotype is still lacking a depth of mechanistic insights needed to assist much needed ability to forecast mutational fates. Here, we address this issue by culminating high throughput mutational scanning using deep sequencing. This approach allowed comprehensive mechanistic investigation of environmental effects on molecular evolution. We monitored effects of various physical and chemical environments onto single site mutants of model antibiotic resistant gene. Alongside, to get mechanistic understanding, we identified multiple molecular constraints which contribute to various degrees in determining the resulting survivabilities of mutants. Across all tested environments, we find that along with substrate binding, protein stability stands out as the common strong constraints. Remarkable direct dependence of the environmental fitness effects on the type of environmental alteration of protein folding further proves that protein stability is the major constraint of the gene. So, our findings reveal that under the influence of environmental conditions, mutational fates are channeled by various degrees of strengths of underlying molecular constraints.