Structural features and the persistence of acquired proteins.

ORFan genes can constitute a large fraction of a bacterial genome, but due to their lack of homologs, their functions have remained largely unexplored. To determine if particular features of ORFan-encoded proteins promote their presence in a genome, we analyzed properties of ORFans that originated over a broad evolutionary timescale. We also compared ORFan genes to another class of acquired genes, heterogeneous occurrence in prokaryotes (HOPs), which have homologs in other bacteria. A total of 54 ORFan and HOP genes selected from different phylogenetic depths in the Escherichia coli lineage were cloned, expressed, purified, and subjected to circular dichroism (CD) spectroscopy. A majority of genes could be expressed, but only 18 yielded sufficient soluble protein for spectral analysis. Of these, half were significantly alpha-helical, three were predominantly beta-sheet, and six were of intermediate/indeterminate structure. Although a higher proportion of HOPs yielded soluble proteins with resolvable secondary structures, ORFans resembled HOPs with regard to most of the other features tested. Overall, we found that those ORFan and HOP genes that have persisted in the E. coli lineage were more likely to encode soluble and folded proteins, more likely to display environmental modulation of their gene expression, and by extrapolation, are more likely to be functional.