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The kinetics of refolding of Escherichia coli dihydrofolate reductase (EC 1.5.1.3) have been examined upon dilution of unfolded enzyme in 4.5 M urea to 1.29 M urea in 0.02 M phosphate buffer (pH 7.2) at 10 degrees C. Changes in the intrinsic protein fluorescence on refolding are characterized by four phases. Based on changes in the amplitudes of these phases, as a consequence of quenching of the intrinsic fluorescence by ligands, it is possible to determine the step at which a ligand binds during the refolding process. The results show that either NADP or NADPH binds to the last species formed in a sequence involving three intermediates between the unfolded and native states. Dihydrofolate, on the other hand, binds during the formation of the second observed intermediate. When refolding is performed in the presence of methotrexate, an analogue of dihydrofolate, and NADPH, NADPH binds, as determined from changes in NADPH fluorescence, to the third observed intermediate rather than the last (fourth) species formed. Measurements of the recovery of enzymatic activity during refolding suggest that dihydrofolate also induces NADPH binding prior to the final observed folding phase. These results define more closely the formation of structural domains during the folding of dihydrofolate reductase.

Refolding of Escherichia coli dihydrofolate reductase: sequential formation of substrate binding sites.