Novel folded protein domains generated by combinatorial shuffling of polypeptide segments

It has been proposed that the architecture of protein domains has evolved b y the combinatorial assembly and/or exchange of smaller polypeptide segment s. To investigate this proposal, we fused DNA encoding the N-terminal half of a beta-barrel domain (from cold shock protein CspA) with fragmented geno mic Escherichia coil DNA and cloned the repertoire of chimeric polypeptides for display on filamentous bacteriophage. Phage displaying folded polypept ides were selected by proteolysis; in most cases the protease-resistant chi meric polypeptides comprised genomic segments in their natural reading fram es. Although the genomic segments appeared to have no sequence homologies w ith CspA, one of the originating proteins had the same fold as CspA, but an other had a different fold. Four of the chimeric proteins were expressed as soluble polypeptides; they formed monomers and exhibited cooperative unfol ding. Indeed, one of the chimeric proteins contained a set of very slowly e xchanging amides and proved more stable than CspA itself. These results ind icate that native-like proteins can be generated directly by combinatorial segment assembly from nonhomologous proteins, with implications for theorie s of the evolution of new protein folds, as well as providing a means of cr eating novel domains and architectures in vitro.