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Proteins from an unevolved library of de novo designed sequences bind a range of small molecules.

ABSTRACT: The availability of large collections of de novo designed proteins presents new opportunities to harness novel macromolecules for synthetic biological functions. Many of these new functions will require binding to small molecules. Is the ability to bind small molecules a property that arises only in response to biological selection or computational design? Or alternatively, is small molecule binding a property of folded proteins that occurs readily amidst collections of unevolved sequences? These questions can be addressed by assessing the binding potential of de novo proteins that are designed to fold into stable structures, but are "naïve" in the sense that they (i) share no significant sequence similarity with natural proteins and (ii) were neither selected nor designed to bind small molecules. We chose three naïve proteins from a library of sequences designed to fold into 4-helix bundles and screened for binding to 10,000 compounds displayed on small molecule microarrays. Several binders were identified, and binding was characterized by a series of biophysical assays. Surprisingly, despite the similarity of the three de novo proteins to one another, they exhibit selective ligand binding. These findings demonstrate the potential of novel proteins for molecular recognition and have significant implications for a range of applications in synthetic biology.

SUBMITTER: Cherny I

PROVIDER: S-EPMC4104770 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Proteins from an unevolved library of de novo designed sequences bind a range of small molecules.

Cherny Izhack I Korolev Maria M Koehler Angela N AN Hecht Michael H MH

ACS synthetic biology 20120402 4

The availability of large collections of de novo designed proteins presents new opportunities to harness novel macromolecules for synthetic biological functions. Many of these new functions will require binding to small molecules. Is the ability to bind small molecules a property that arises only in response to biological selection or computational design? Or alternatively, is small molecule binding a property of folded proteins that occurs readily amidst collections of unevolved sequences? Thes ...[more]

PMID: 23651114

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Project description:Combinatorial libraries of de novo amino acid sequences can provide a rich source of diversity for the discovery of novel proteins. Randomly generated sequences, however, rarely fold into well ordered protein-like structures. To enhance the quality of a library, diversity must be focused into those regions of sequence space most likely to yield well folded structures. We have constructed focused libraries of de novo sequences by designing the binary pattern of polar and nonpolar amino acids to favor structures that contain abundant secondary structure, while simultaneously burying hydrophobic side chains in the protein interior and exposing hydrophilic side chains to solvent. Because binary patterning specifies only the polar/nonpolar periodicity, but not the identities of the side chains, detailed structural features, including packing interactions, cannot be designed a priori. Can binary patterned libraries nonetheless encode well folded proteins? An unambiguous answer to this question requires determination of a 3D structure. We used NMR spectroscopy to determine the structure of S-824, a novel protein from a recently constructed library of 102-residue sequences. This library is "naïve" in that it has not been subjected to high-throughput screens or directed evolution. The experimentally determined structure of S-824 is a four-helix bundle, as specified by the design. As dictated by the binary-code strategy, nonpolar side chains are buried in the protein interior, and polar side chains are exposed to solvent. The polypeptide backbone and buried side chains are well ordered, demonstrating that S-824 is not a molten globule and forms a unique structure. These results show that amino acid sequences that have neither been selected by evolution, nor designed by computer, nor isolated by high-throughput screening, can form native-like structures. These findings validate the binary-code strategy as an effective method for producing vast collections of well folded de novo proteins.

Dataset Information

Proteins from an unevolved library of de novo designed sequences bind a range of small molecules.

Publications

Proteins from an unevolved library of de novo designed sequences bind a range of small molecules.

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