Reverse engineering the (beta/alpha )8 barrel fold.

The (beta/alpha)(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 (beta/alpha)(8) barrel enzyme triosephosphate isomerase. Using combinatorial mutagenesis and functional selection, we find that turn sequences, alpha-helix capping and stop motifs, and residues that pack the interface between beta-strands and alpha-helices are highly mutable. Conversely, any mutation of residues in the central core of the beta-barrel, beta-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 (beta/alpha)(8) barrel structures can be encoded with a subset of the 20 amino acids. Such simplification would greatly decrease the computational burden of (beta/alpha)(8) barrel design.