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Stably folded de novo proteins from a designed combinatorial library

Department of Chemistry, Princeton University, Princeton, New Jersey 08544-1009, USA

Reprint requests to: Michael H. Hecht, Department of Chemistry, Princeton University, Princeton, NJ 08544-1009, USA; e-mail: hecht{at} princeton.edu; fax: (609) 258-6746.

Binary patterning of polar and nonpolar amino acids has been used as the key design feature for constructing large combinatorial libraries of de novo proteins. Each position in a binary patterned sequence is designed explicitly to be either polar or nonpolar; however, the precise identities of these amino acids are varied extensively. The combinatorial underpinnings of the "binary code" strategy preclude explicit design of particular side chains at specified positions. Therefore, packing interactions cannot be specified a priori. To assess whether the binary code strategy can nonetheless produce well-folded de novo proteins, we constructed a second-generation library based upon a new structural scaffold designed to fold into 102-residue four-helix bundles. Characterization of five proteins chosen arbitrarily from this new library revealed that (1) all are -helical and quite stable; (2) four of the five contain an abundance of tertiary interactions indicative of well-ordered structures; and (3) one protein forms a well-folded structure with native-like features. The proteins from this new 102-residue library are substantially more stable and dramatically more native-like than those from an earlier binary patterned library of 74-residue sequences. These findings demonstrate that chain length is a crucial determinant of structural order in libraries of de novo four-helix bundles. Moreover, these results show that the binary code strategy—if applied to an appropriately designed structural scaffold—can generate large collections of stably folded and/or native-like proteins.

Keywords: Protein design; binary patterning; de novo proteins; native-like protein structure; combinatorial library; four-helix bundle

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