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Cofactor binding and enzymatic activity in an unevolved superfamily of de novo designed 4-helix bundle proteins†

  1. Shona C. Patel1,‡,
  2. Luke H. Bradley2,§,
  3. Sayuri P. Jinadasa2,¶,
  4. Michael H. Hecht2,*

Article first published online: 29 APR 2009

DOI: 10.1002/pro.147

Issue

Protein Science

Protein Science

Volume 18, Issue 7, pages 1388–1400, July 2009

Additional Information

How to Cite

Patel, S. C., Bradley, L. H., Jinadasa, S. P. and Hecht, M. H. (2009), Cofactor binding and enzymatic activity in an unevolved superfamily of de novo designed 4-helix bundle proteins. Protein Science, 18: 1388–1400. doi: 10.1002/pro.147

Author Information

  1. 1

    Department of Chemical Engineering, Princeton University, Princeton, New Jersey 08544

  2. 2

    Department of Chemistry, Princeton University, Princeton, New Jersey 08544

  1. ‡

    Merck & Co., Inc., West Point, PA 19486

  2. §

    Departments of Anatomy & Neurobiology, Molecular & Cellular Biochemistry, University of Kentucky, Lexington, KY 40536

  3. Columbia University Medical Center, New York, NY 10032

*Department of Chemistry, Princeton University, Princeton, NJ, 08544

  1. †

    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.

Publication History

  1. Issue published online: 23 JUN 2009
  2. Article first published online: 29 APR 2009
  3. Accepted manuscript online: 29 APR 2009 12:00AM EST
  4. Manuscript Accepted: 13 APR 2009
  5. Manuscript Revised: 12 APR 2009
  6. Manuscript Received: 1 MAR 2009

Funded by

  • National Science Foundation Graduate Fellowship
  • Princeton University Council on Science and Technology
  • NSF grant. Grant Number: MCB-0817651

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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.

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