TOPOLOGICAL MUTATION OF ESCHERICHIA-COLI DIHYDROFOLATE-REDUCTASE

Proteins appear to contain structural elements which determine the fol ded structure. If such elements are present, the order of structural e lements in the primary structure, i,e,the chain topology, can be disre garded for building of the folded tertiary structure, when they are pr operly connected to each other by proper linkers. To experimentally ex amine this, ''topological'' mutants (designated as GHF33 and GHF34) of Escherichia cold dihydrofolate reductase (DHFR) were designed and con structed by switching two amino acid sequence parts containing the bet a 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 b ar>, was changed to beta A --> alpha B --> beta B --> alpha C --> beta C --> beta D --> alpha E --> beta E --> alpha F --> <(beta G)under ba r> --> <(beta H)under bar> --> <(beta F)under bar>. Such topological m utant proteins were stably expressed and accumulated in E. coli cells, and highly purified. Molecular mass measurements of the purified prot eins and their proteolytic fragments confirmed that GHF33 and GHF34 ha d the designed sequence. In terms of k(cat), the GHF33 and GHF34 prote ins 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. Th e topological mutants showed a cooperative two-state transition in ure a-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 DHF R were 0.9 mu M and 6.1 kcal/mol, respectively. The significance of th e topological mutations was discussed with respect to protein folding and protein evolution.