Molecular ensembles make evolution unpredictable.

Evolutionary prediction is of deep practical and philosophical importance. Here we show, using a simple computational protein model, that protein evolution remains unpredictable, even if one knows the effects of all mutations in an ancestral protein background. We performed a virtual deep mutational scan-revealing the individual and pairwise epistatic effects of every mutation to our model protein-and then used this information to predict evolutionary trajectories. Our predictions were poor. This is a consequence of statistical thermodynamics. Proteins exist as ensembles of similar conformations. The effect of a mutation depends on the relative probabilities of conformations in the ensemble, which in turn, depend on the exact amino acid sequence of the protein. Accumulating substitutions alter the relative probabilities of conformations, thereby changing the effects of future mutations. This manifests itself as subtle but pervasive high-order epistasis. Uncertainty in the effect of each mutation accumulates and undermines prediction. Because conformational ensembles are an inevitable feature of proteins, this is likely universal.