High-Resolution Crystal Structure of an Artificial (βα)8-Barrel Protein Designed from Identical Half-Barrels

Ample evidence suggests that the ubiquitous (beta alpha)(8)-barrel enzyme fold has evolved by the duplication and fusion of an ancestral (beta alpha)(4)-half-barrel. To reconstruct this process in the laboratory with a model protein, we earlier fused two copies of the C-terminal half-barrel HisF-C of imidazole glycerol phosphate synthase, (HisF) and stepwise stabilized the resulting HisF-CC construct. We now further increased its stability and Solubility by introducing two additional amino acid exchanges, which allowed us to crystallize the resulting artificial (beta alpha)(8)-barrel protein HisF-C***C. The analysis of its X-ray structure at 2.1 angstrom resolution reveals a striking similarity to wildtype HisF, helps us to understand its improved stability, and provides further insights into the evolution of (beta alpha)(8)-barrel proteins.