Artificial duplication of the R67 dihydrofolate reductase gene to create protein asymmetry. Effects on protein activity and folding.
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 DHFRdouble, is twice the molecular mass of native R67 DHFR and is fully active with kcat = 1.2 s-1, Km(NADPH) = 2.7 microM and Km(dihydrofolate) = 6.3 microM. Equilibrium unfolding studies in guanidine-HCl indicate R67 DHFRdouble 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 delta Hcalorimetric to delta Hvan't Hoff equals 0.96. In contrast, thermal unfolding of R67 DHFRdouble is not fully reversible and possesses an oligomerization component introduced by the gene duplication event.
Zhuang
P
P
Department of Biochemistry, University of Tennessee, Knoxville 37996-0840.
Yin
M
M
Holland
J C
JC
Peterson
C B
CB
Howell
E E
EE
D000595:Amino Acid Sequence; D001483:Base Sequence; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D005798:Genes, Bacterial; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008961:Models, Structural; D008969:Molecular Sequence Data; D005810:Multigene Family; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011815:R Factors; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase