Abstract:

The potential for enzymatic activity in unselected amino acid sequence space was probed by creating a combinatorial library of de novo four-helix bundle proteins. The collection of 102-residue proteins, regarded as a de novo superfamily, was designed using binary patterning of polar and nonpolar residues, and expressed in E. coli from a library of synthetic genes. These de novo designed, four-helix bundles were screened for a range of biological functions, including heme binding, and peroxidase, esterase and lipase activities. Proteins exhibiting these functions were purified and characterized biochemically. Finally, the potential for directed evolution was explored and high-throughput screening methods were implemented for peroxidase and esterase activity.

The majority of de novo proteins from this superfamily bound the heme cofactor, and many proteins showed activity significantly above background for either peroxidase, esterase or lipase activity. Moreover, several of the designed four-helix bundles proteins showed activity in all of the assays, thereby demonstrating the functional promiscuity of unevolved proteins. These studies demonstrate that de novo proteins--which have neither been designed for function, nor subjected to evolutionary pressure--can provide rudimentary activities and serve as a "feedstock" for evolution.