Exploring protein fitness landscapes by directed evolution

Key Points

  • Directed evolution optimizes protein function by the successive generations of random mutation, artificial selection or screening. This simple design algorithm circumvents our ignorance of how sequence encodes function and provides a reliable approach to engineering proteins with new and useful properties.

  • Directed evolution can be envisioned as an uphill walk on a protein fitness landscape, in which regions of higher elevation represent more optimized proteins. The ruggedness of this fitness landscape affects the ability to find uphill paths to fitter sequences and therefore affects the ease of evolutionary searches.

  • Simple adaptive walks effectively optimize many protein functions, despite landscape ruggedness that arises from epistatic interactions between mutations. The many simple uphill routes to higher fitness can circumvent more convoluted paths that involve neutral or deleterious mutations. More-stable proteins can accept a wider ranger of mutations and are more evolvable.

  • Recombination of homologous protein sequences provides access to functional sequences with many mutations. These recombined (chimeric) proteins can exhibit properties outside the range of the parental sequences, such as higher stability or even novel activities.

  • Directed evolution studies have generated a wealth of information on the structure of protein fitness landscapes, mechanisms of adaption, pathways that are accessible under different selection pressures and the nature of trade-offs between properties during evolution.

Abstract

Directed evolution circumvents our profound ignorance of how a protein's sequence encodes its function by using iterative rounds of random mutation and artificial selection to discover new and useful proteins. Proteins can be tunedto adapt to new functions or environments by simple adaptive walks involving small numbers of mutations. Directed evolution studies have shown how rapidly some proteins can evolve under strong selection pressures and, because the entire 'fossil record' of evolutionary intermediates is available for detailed study, they have provided new insight into the relationship between sequence and function. Directed evolution has also shown how mutations that are functionally neutral can set the stage for further adaptation.

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Figure 1: Protein fitness landscapes.
Figure 2: Overview of directed evolution.
Figure 3: Recombination of homologous sequences.
Figure 4: Directed evolution of a cytochrome P450 propane monooxygenase.
Figure 5: Stability threshold and epistasis.

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Acknowledgements

The authors acknowledge support from the U.S. Army Research Office, Department of Energy, National Science Foundation and the National Institutes of Health.

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Glossary

Evolvability

A measure of the ability of a protein to adapt in response to mutation and selective pressure; for example, the frequency of beneficial mutations.

Directed evolution

The application of iterative rounds of mutation and artificial selection or screening to alter the properties of biological molecules and systems

Fitness landscape

The mapping from genotype (target sequence) to phenotype (fitness; as measured in the experiment). Directed evolution is an optimization on the fitness landscape.

Recombination

A procedure whereby chimeric proteins are created by recombining sequence fragments from different (usually evolutionarily, and therefore structurally, related) parent proteins.

Protein sequence space

The space of all possible protein sequences arranged such that sequences that differ by single mutations are neighbours.

Adaptive walk

An uphill trajectory on the fitness landscape, in which no deleterious mutations are accepted.

Neutral drift

The accumulation of mutations that have little or no effect on a particular protein function. These mutations, however, might affect other properties.

Neutral network

An interconnected network of functionally neutral sequences.

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Romero, P., Arnold, F. Exploring protein fitness landscapes by directed evolution. Nat Rev Mol Cell Biol 10, 866–876 (2009). https://doi.org/10.1038/nrm2805

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