Radical redesign of a tandem array of four R67 dihydrofolate reductase genes yields a functional, folded protein possessing 45 substitutions.

R67 dihydrofolate reductase (DHFR) is a plasmid-encoded, type II enzyme. Four monomers (78 amino acids long) assemble into a homotetramer possessing 222 symmetry. In previous studies, a tandem array of four R67 DHFR gene copies was fused in frame to generate a functional monomer named Quad1. This protein possessed the essential tertiary structure of the R67 "parent". To facilitate mutagenesis reactions, restriction enzyme sites were introduced in the tandem gene array. S59A and H362L mutations were also added to minimize possible folding topologies; this protein product, named Quad3, possesses 10 substitutions and is functional. Since R67 DHFR possesses a stable scaffold, a large jump in sequence space was performed by the further addition of 45 amino acid substitutions. The mutational design utilized alternate sequences from other type II DHFRs. In addition, most of the mutations were positioned on the surface of the protein as well as in the disordered N-terminal sequence, which serves as the linker between the fused domains. The resulting Quad4 protein is quite functional; however, it is less stable than Quad1, suffering a DeltaDeltaG loss of 5 kcal/mol at pH 5. One unexpected result was formation of Quad4 dimers and higher order oligomers at pH 8. R67 DHFR, and its derivative Quad proteins, possesses a robust scaffold, capable of withstanding introduction of >or=55 substitutions.