Cofactor binding and enzymatic activity in an unevolved superfamily of de novo designed 4-helix bundle proteins.
To probe the potential for enzymatic activity in unevolved amino acid sequence space, we created a combinatorial library of de novo 4-helix bundle proteins. This collection of novel proteins can be considered an "artificial superfamily" of helical bundles. The superfamily of 102-residue proteins was designed using binary patterning of polar and nonpolar residues, and expressed in Escherichia coli from a library of synthetic genes. Sequences from the library 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. The majority of de novo proteins from this superfamily bound the heme cofactor, and a sizable fraction of the proteins showed activity significantly above background for at least one of the tested enzymatic activities. Moreover, several of the designed 4-helix bundles proteins showed activity in all of the assays, thereby demonstrating the functional promiscuity of unevolved proteins. These studies reveal that de novo proteins-which have neither been designed for function, nor subjected to evolutionary pressure (either in vivo or in vitro)-can provide rudimentary activities and serve as a "feedstock" for evolution.
Patel
Shona C
SC
Department of Chemical Engineering, Princeton University, Princeton, New Jersey 08544, USA.
Bradley
Luke H
LH
Jinadasa
Sayuri P
SP
Hecht
Michael H
MH
D000595:Amino Acid Sequence; D055162:Biocatalysis; D003067:Coenzymes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004798:Enzymes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019143:Evolution, Molecular; D006418:Heme / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D019151:Peptide Library; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D003067:Coenzymes; D004798:Enzymes; D019151:Peptide Library; D011994:Recombinant Proteins; D006418:Heme