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Exploring protein fitness landscapes by directed evolution
Philip A. Romero and Frances H. Arnold
Abstract | Directed evolution circumvents our profound ignorance of how a proteins sequence encodes its function by using iterative rounds of random mutation and artificial selection to discover new and useful proteins. Proteins can be tuned to 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.
Millions of years of lifes struggle for survival in differen t environments have resulted in proteins providing diverse, creative and efficient solutions to a wide range of problems, from extracting energy from the environment to repairing and replicating their own code. Good solutions to biological problems can also be good solutions to human problems proteins are widely used in the food, chemicals, consumer products and medical fields. Not content with natures protein repertoire, however, protein engineers are working to extend known protein function to new environments or tasks14 and to create new functions altogether57.
Despite major advances, a molecular-level understanding of why one protein performs a certain task better than another remains elusive. This is perhaps not surprising when we remember that a protein often undergoes conformational changes during function and exists as a dynamic ensemble of conformers that are only slightly more stable than their unfolded and non-functional states and that might themselves be functionally diverse8. Mutations far away from active sites can influence protein function9,10. Engineering
enzymatic activity is particularly difficult because very small changes in structure or chemical properties can have big effects on catalysis. Thus, predicting the amino acid sequence, or changes to an amino acid sequence, that would generate a specific behaviour remains a challenge, particularly for applications requiring high performance (such as an industrial enzyme or a therapeutic protein). Unfortunately, where function is concerned, details matter, and we just dont understand the details.
Evolution, however, had no difficulty generating these impressive molecules....