Radical Redesign of a Tandem Array of Four R67 Dihydrofolate Reductase Genes Yields a Functional, Folded Protein Possessing 45 Substitutions

摘要

R67 dihydrofolate reductase (DHFR) is a plasmid-encoded, type II enzyme. Four monomersn(78 amino acids long) assemble into a homotetramer possessing 222 symmetry. In previous studies, a tandemnarray of four R67 DHFR gene copies was fused in frame to generate a functional monomer named Quad1.nThis protein possessed the essential tertiary structure of theR67 “parent”. To facilitatemutagenesis reactions,nrestriction enzyme sites were introduced in the tandem gene array. S59A and H362L mutations were alsonadded tominimize possible folding topologies; this protein product, named Quad3, possesses 10 substitutionsnand is functional. Since R67DHFR possesses a stable scaffold, a large jump in sequence space was performednby the further addition of 45 amino acid substitutions. The mutational design utilized alternate sequencesnfromother type IIDHFRs. In addition,most of themutations were positioned on the surface of the protein asnwell as in the disordered N-terminal sequence, which serves as the linker between the fused domains. Thenresulting Quad4 protein is quite functional; however, it is less stable than Quad1, suffering a ΔΔG loss ofn5 kcal/mol at pH 5. One unexpected result was formation of Quad4 dimers and higher order oligomers at pH 8.nR67 DHFR, and its derivative Quad proteins, possesses a robust scaffold, capable of withstanding intro-nduction of ≥55 substitutions.