Reverse engineering the (bya)8 barrel fold

The (bya)8 barrel is the most commonly occurring fold among protein catalysts. To lay a groundwork for engineering novel barrel proteins, we investigated the amino acid sequence restrictions at 182 structural positions of the prototypical (bya)8 barrel enzyme triosephosphate isomerase. Using combinatorial mutagenesis and functional selection, we find that turn sequences, a-helix capping and stop motifs, and residues that pack the interface between b-strands and a-helices are highly mutable. Conversely, any mutation of residues in the central core of the b-barrel, b-strand stop motifs, and a single buried salt bridge between amino acids R189 and D227 substantially reduces catalytic activity. Four positions are effectively immutable: conservative single substitutions at these four positions prevent the mutant protein from complementing a triosephosphate isomerase knockout in Escherichia coli. At 142 of the 182 positions, mutation to at least one amino acid of a seven-letter amino acid alphabet produces a triosephosphate isomerase with wild-type activity. Consequently, it seems likely that (bya)8 barrel structures can be encoded with a subset of the 20 amino acids. Such simplification would greatly decrease the computational burden of (bya)8 barrel design.