Article
The authors dedicate this article to the memory of Walter Kauzmann, a long time member of the Princeton Department of Chemistry. His early insights into the importance of the hydrophobic effect in protein folding were decades ahead of their time, and laid the foundation for the protein design strategy described in this article.
Received: 1 March 2009; Revised: 12 April 2009; Accepted: 13 April 2009
10.1002/pro.147 About DOI
Cofactor binding and enzymatic activity in an unevolved superfamily of de novo designed 4-helix bundle proteins |
| Shona C. Patel 1 a, Luke H. Bradley 2 b, Sayuri P. Jinadasa 2 c, Michael H. Hecht 2 * |
| 1Department of Chemical Engineering, Princeton University, Princeton, New Jersey 08544 2Department of Chemistry, Princeton University, Princeton, New Jersey 08544 |
| email: Michael H. Hecht (hecht@princeton.edu) |
*Correspondence to Michael H. Hecht, Department of Chemistry, Princeton University, Princeton, NJ, 08544
aCurrent address: Merck & Co., Inc., West Point, PA 19486
bCurrent address: Departments of Anatomy & Neurobiology, Molecular & Cellular Biochemistry, University of Kentucky, Lexington, KY 40536
cCurrent address: Columbia University Medical Center, New York, NY 10032
The authors dedicate this article to the memory of Walter Kauzmann, a long time member of the Princeton Department of Chemistry. His early insights into the importance of the hydrophobic effect in protein folding were decades ahead of their time, and laid the foundation for the protein design strategy described in this article.Funded by:
National Science Foundation Graduate Fellowship Princeton University Council on Science and Technology NSF grant; Grant Number: MCB-0817651
| Keywords |
| binary code protein design biomolecular evolution 4-helix bundle synthetic biology |
| Abstract |
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. |
Received: 1 March 2009; Revised: 12 April 2009; Accepted: 13 April 2009
| Digital Object Identifier (DOI) |
10.1002/pro.147 About DOI
