Selecting folded proteins from a library of secondary structural elements

Despite the large sequence diversity present in the proteomes of known organisms, most functional proteins characterized to date assume one of relatively few distinct folds1-5, suggesting that nature uses a limited number of stable, soluble folds relative to what is theoretically possible in protein sequence space. These folds likely represent the divergent products of a limited set of ancient protein folds. However, protein sequences that fold into stable, unique topologies but are not encoded by any sequenced genome may also exist. It may be possible to identify other stable folds that simply have not yet been sampled in the course of evolution by means of an artificial selection and molecular evolution process. Methods such as DNA shuffling6 which mimic the combinatorial diversity mechanism of the immune system are among the most efficient methods to modify or enhance protein activity. Unfortunately, the structural diversity generated in a library of shuffled homologues is relatively small7-11, although newer methods for random fragment assembly that overcome sequence homology requirements may lead to libraries with increased structural diversity12,13. Alternatively, natural or in vitro combinatorial assembly of distinct protein subunits (e.g., subdomains, exons, etc.) can create significant structural and functional diversity14-19. For example, the mammalian blood clotting proteins plasminogen, protein C, urokinase and prothrombin are all derived from different combinations of 5 exons14. Sequence and structural studies also suggest that many existing TIM barrel proteins evolved from the combinatorial assembly of subunits from more primitive eightfold βα barrels15,16. Other approaches for creating libraries of novel sequences that fold into native-like protein structures include the use of binary patterned residues designed around an existing known fold and the random assembly of cassettes composed of as few as three amino acid residues17,18. A more recent report details the use of cassettes of binary patterned residues around known protein motifs that are ultimately randomly assembled without fitting any designed folding constraint19. However, these latter methods have not yet yielded stable, novel folds under physiological conditions20. Despite the considerable effort to identify or evolve new folds, only a limited number of distinct protein folds have been identified to date18. Here we describe a system for the synthesis of polypeptides with potentially novel folds in which existing sequence and structural data from bacterial proteins are used to assemble a pool of secondary structural elements (α-helices, β-strands and loops). These secondary structural elements are in turn recombined into a library of de novo sequences that may be screened or selected for folded proteins. Proteins that fold into a native state acquire measurable biophysical characteristics such as solubility, stable secondary structure, well-dispersed 1D NMR spectra indicative of packed side chains, and reversible two-state unfolding behavior. In this work, we chose to initially screen the library for polypeptides that are soluble in aqueous solution using a GFP fusion protein reporter that had been previously reported21. Indeed, several sequences with stable secondary structure are identified.