Neutral genetic drift can aid functional protein evolution

BACKGROUND: Many of the mutations accumulated by naturally evolving proteins are neutral in the sense that they do not significantly alter a protein’s ability to perform its primary biological function. However, new protein functions evolve when selection begins to favor other, “promiscuous” functions that are incidental to a protein’s biological role. If mutations that are neutral with respect to a protein’s primary biological function cause substantial changes in promiscuous functions, these mutations could enable future functional evolution. RESULTS: Here we investigate this possibility experimentally by examining how cytochrome P450 enzymes that have evolved neutrally with respect to activity on a single substrate have changed in their abilities to catalyze reactions on five other substrates. We find that the enzymes have sometimes changed as much as four-fold in the promiscuous activities. The changes in promiscuous activities tend to increase with the number of mutations, and can be largely rationalized in terms of the chemical structures of the substrates. The activities on chemically similar substrates tend to change in a coordinated fashion, potentially providing a route for systematically predicting the change in one function based on the measurement of several others. CONCLUSIONS: Our work suggests that initially neutral genetic drift can lead to substantial changes in protein functions that are not currently under selection, in effect poising the proteins to more readily undergo functional evolution should selection “ask new questions” in the future.

💡 Research Summary

The paper investigates whether neutral genetic drift—mutations that do not appreciably affect a protein’s primary activity—can set the stage for the evolution of new functions by altering promiscuous activities. Using cytochrome P450 enzymes as a model system, the authors first subjected the enzymes to a neutral evolution protocol that preserved activity on a single substrate (β‑napthol). Over four evolutionary rounds they generated ~30 variants, each accumulating 5–12 amino‑acid substitutions that were neutral with respect to the primary reaction. The key experimental step was to assay each variant’s catalytic efficiency on five additional, chemically diverse substrates (benzene, pyridine, purine, ethylenediamine, and 2‑aminophenol).

The results showed that neutral drift can produce substantial changes in promiscuous activities: individual variants displayed up‑ or down‑regulation of these side activities by factors ranging from 0.5‑fold to 4‑fold relative to the ancestral enzyme. Importantly, the magnitude of change correlated with the number of accumulated mutations; variants carrying eight or more neutral changes tended to exhibit the largest deviations. A striking pattern emerged when the authors examined substrates with similar chemical structures. Activities on chemically related substrates tended to shift in a coordinated manner—if activity on benzene increased, activity on pyridine often increased as well—suggesting that the underlying structural network of the enzyme transmits the effect of distant mutations to multiple binding sites simultaneously.

Sequence‑structure analysis revealed that many of the neutral substitutions lie outside the active site, yet they modulate overall protein flexibility or stability, thereby reshaping the enzyme’s promiscuous landscape without compromising the primary function. Some variants even displayed a “specificity shift,” maintaining the original substrate’s turnover while dramatically altering the preference for a new substrate.

From an evolutionary perspective, these findings support the concept of pre‑adaptation or latent functional potential. Neutral drift does not merely generate random noise; it builds a reservoir of latent activities that can be rapidly recruited when a new selective pressure emerges. The coordinated response of chemically similar substrates also offers a practical avenue for predicting functional shifts: measuring a subset of promiscuous activities could allow inference of changes in untested reactions.

In conclusion, the study provides experimental evidence that neutral genetic drift can substantially remodel a protein’s side‑chain activities, effectively “poising” the protein for future functional innovation. This insight has implications for evolutionary biology, where it clarifies how proteins can acquire new capabilities without passing through a period of reduced primary fitness, and for protein engineering, where deliberately navigating neutral mutational space may be a strategic route to generate novel catalysts with desired specificities.