EQUILIBRIUM FOLDING STUDIES OF TETRAMERIC R67 DIHYDROFOLATE-REDUCTASE

R67 dihydrofolate reductase (DHFR) is an R-plasmid encoded enzyme that confers resistance to the antibacterial drug trimethoprim. This enzym e is not homologous in sequence or structure to chromosomal DHFRs. Equ ilibrium folding of tetrameric R67 DHFR was studied and found to be fu lly reversible. Formation of an inactive intermediate was assayed by l oss of enzyme activity. Denaturation of the intermediate was monitored by concurrent changes in fluorescence and circular dichroism signals. Both transitions are protein concentration dependent. A simple model fitting these data is tetramer reversible 2 dimers reversible 4 unfold ed monomers. No evidence for folded monomer was found. Global fitting of all the folding data yielded a DELTAG(H2O) of -9.63 kcal/mol for th e initial transition and a DELTAG(H2O) of -12.35 kcal/mol for the seco nd transition. In addition, thermal unfolding of tetrameric R67 DHFR w as found to be reversible. A folding intermediate also occurred during thermal unfolding as evidenced by the asymmetric endotherms and a DEL TAH(calorimetric)/DELTAH(van't Hoff) ratio of 2.1.