Artificial duplication of the R67 dihydrofolate reductase gene to create protein asymmetry. Effects on protein activity and folding
Abstract
R67 dihydrofolate reductase (DHFR), encoded by an R plasmid, provides resistance to the antibacterial drug trimethoprim. This enzyme does not exhibit any structural or sequence homologies with chromosomal DHFR. A recent crystal structure of tetrameric R67 DHFR (D. Matthews, X. Nguyen-huu, and N. Narayana, personal communication) shows a single pore traversing the length of the molecule. Numerous physical and kinetic experiments suggest the pore is the active site. Since the center of the pore possesses exact 222 symmetry, mutagenesis of residues designed to explore substrate binding will probably also affect cofactor binding. As a first step in breaking this inevitable symmetry in R67 DHFR, the gene has been duplicated. The protein product, R67 DHFR(double), is twice the molecular mass of native R67 DHFR and is fully active with k(cat) = 1.2 s-1, K(m)((NADPH)) = 2.7 μM and K(m)((dihydrofolate)) = 6.3 μM. Equilibrium unfolding studies in guanidine- HCl indicate R67 DHFR(double) is more stable than native R67 DHFR at physically reasonable protein concentrations. Microcalorimetry studies show native R67 DHFR undergoes fully reversible thermal unfolding. Unfolding can be described by a two-state process since a ratio of ΔH(calorimetric) to ΔH(van't Hoff) equals 0.96. In contrast, thermal unfolding of R67 DHFR(double) is not fully reversible and possesses an oligomerization component introduced by the gene duplication event.