Directed evolution of the peroxidase activity of a de novo-designed protein.

Collections of de novo-designed proteins provide a unique opportunity to probe the functional potential of sequences that are stably folded, but were neither explicitly designed nor evolutionarily selected to perform any particular type of activity. A combinatorial library of folded proteins was designed previously using a strategy that exploits the binary patterning of polar and non-polar amino acids to drive sequences to fold into four-helix bundles. Although these novel four-helix bundles were not explicitly designed for function, previous characterization of several hundred arbitrarily chosen sequences showed that many of them bound the heme cofactor, and several of these novel heme proteins catalyzed peroxidase activity at levels substantially above background. Here, we show that these moderately active de novo heme proteins can serve as non-natural starting points for laboratory-based evolution: Random mutagenesis followed by color-based screening of a relatively small number (hundreds or thousands) of variants yielded novel sequences with improved peroxidase activity. Biochemical characterization of the purified proteins showed that the evolved variants were nearly 3-fold more active than the parental sequence. These results demonstrate that de novo-designed proteins can be utilized as a novel feedstock for the evolution of enzyme activity.