Topological mutation of Escherichia coli dihydrofolate reductase

Proteins appear to contain structural elements which determine the folded structure. If such elements are present, the order of structural elements in the primary structure, i,e,the chain topology, can be disregarded for building of the folded tertiary structure, when they are properly connected to each other by proper linkers. To experimentally examine this, "topological" mutants (designated as GHF33 and GHF34) of Escherichia cold dihydrofolate reductase (DHFR) were designed and constructed by switching two amino acid sequence parts containing the beta F strand and beta G-beta H strands in the primary sequence. In this way, the chain topology of wild-type DHFR, beta A --> alpha B --> beta B --> alpha C --> beta C --> beta D --> alpha E --> beta E --> alpha F --> <(beta F)under bar> --> <(beta G)under bar> --> <(beta H)under bar>, was changed to beta A --> alpha B --> beta B --> alpha C --> beta C --> beta D --> alpha E --> beta E --> alpha F --> <(beta G)under bar> --> <(beta H)under bar> --> <(beta F)under bar>. Such topological mutant proteins were stably expressed and accumulated in E. coli cells, and highly purified. Molecular mass measurements of the purified proteins and their proteolytic fragments confirmed that GHF33 and GHF34 had the designed sequence. In terms of k(cat), the GHF33 and GHF34 proteins showed about 10 and 20% of the DHFR activity of the wild-type with K-m values of 3.3 mu M (GHF33) and 2.1 mu M (GHF34), respectively. The topological mutants showed a cooperative two-state transition in urea-induced unfolding experiments with Delta G(H2O) values of 4.0 and 4.8 kcal/mol. Whereas, the K-m and Delta G(H2O) values for wild-type DHFR were 0.9 mu M and 6.1 kcal/mol, respectively. The significance of the topological mutations was discussed with respect to protein folding and protein evolution.