Effects of single-tryptophan mutations on R67 dihydrofolate reductase.
R67 dihydrofolate reductase (DHFR) is an R-plasmid-encoded enzyme that confers clinical resistance to the antibacterial drug trimethoprim. This enzyme shows no sequence or structural homology to the chromosomal DHFRs. The active form of the protein is a homotetramer possessing D(2) symmetry and a single active-site pore. Two tryptophans occur per monomer: W38 and its symmetry-related residues (W138, W238, and W338) occur at the dimer-dimer interfaces, while W45 and its symmetry-related partners (W145, W245, and W345) occur at the monomer-monomer interfaces. Two single-tryptophan mutant genes were constructed to determine the structural and functional consequences of four mutations per tetramer. The W45F mutant retains full enzyme activity and the fluorescence environment of the unmutated W38 residues clearly monitors ligand binding and a pH dependent tetramer right harpoon over left harpoon 2 dimers equilibrium. In contrast, four simultaneous W38F mutations at the dimer-dimer interfaces result in tetramer destabilization. The ensuing dimer is relatively inactive, as is dimeric wild-type R67 DHFR. A comparison of emission spectra 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.
West
F W
FW
Department of Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville, 37996-0840, USA.
Seo
H S
HS
Bradrick
T D
TD
Howell
E E
EE
D001665:Binding Sites / Q000235:genetics; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004735:Energy Transfer; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D010649:Phenylalanine / Q000235:genetics; D017510:Protein Folding; D011789:Quantum Theory; D011815:R Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D010649:Phenylalanine; D009249:NADP; D014364:Tryptophan; D013762:Tetrahydrofolate Dehydrogenase