Effects of single-tryptophan mutations on R67 dihydrofolate reductase

R67 dihydrofolate reductase (DHFR) is an R-plasmid-encoded enzyme that conf ers clinical resistance to the ant;bacterial drug trimethoprim. This enzyme shows no sequence or structural homology to the chromosomal DHFRs. The act ive form of the protein is a homotetramer possessing D-2 symmetry and a sin gle active-site pen. Two tryptophans occur per monomer: W38 and its symmetr y-related residues (W138, W238, and W338) occur at the dimer-dimer interfac es, while W45 and its symmetry-related partners (W145, W245, and W345) occu r at the monomer-monomer interfaces. Two single-tryptophan mutant genes wer e constructed to determine the structural and functional consequences of fo ur mutations per tetramer. The W45F mutant retains full enzyme activity and the fluorescence environment of the unmutated W38 residues clearly monitor s ligand binding and a pH dependent tetramer reversible arrow 2 dimers equi librium. In contrast, four simultaneous W38F mutations at the dimer-dimer i nterfaces result in tetramer destabilization. The ensuing dimer is relative ly inactive, as is dimeric wild-type R67 DHFR. A comparison of emission spe ctra indicates the fluorescent signal of wild-type R67 DHFR is dominated by the contribution from W38. Equilibrium unfolding/folding curves at pH 5.0, where all protein variants are dimeric, indicate the environment monitored by the W38 residue is slightly less stable than the environment monitored by the W45 residue.