Redesigning the quaternary structure of R67 dihydrofolate reductase. Creation of an active monomer from a tetrameric protein by quadruplication of the gene.
R67 dihydrofolate reductase (DHFR) provides resistance to the antibacterial drug trimethoprim. This R-plasmid-encoded enzyme does not share any homology with chromosomal DHFR. A recent crystal structure of active, homotetrameric R67 DHFR (Narayana, N., Matthews, D. A., Howell, E. E., and Xuong, N.-H. (1995) Nat. Struct. Biol. 2, 1018-1025) indicates that a single active site pore traverses the length of the molecule. Since the center of the pore possesses exact 222 symmetry, site-directed mutagenesis of residues in the pore will produce four mutations/active site. To break this inevitable symmetry, four copies of the gene have been linked in frame to create an active monomer possessing the essential tertiary structure of native tetrameric R67 DHFR. The protein product, quadruple R67 DHFR, is 4 times the molecular mass of native R67 DHFR in SDS-polyacrylamide gel electrophoresis and is monomeric under nondenaturing conditions as measured by sedimentation equilibrium experiments. The catalytic activity of quadruple R67 DHFR is decreased only slightly when compared with native R67 DHFR. Folding of quadruple R67 DHFR is completely reversible at pH 5. However, at pH 8, folding is not fully reversible; this is likely due to a competition between productive intramolecular versus nonproductive intermolecular domain association. The production of a fully active, monomeric R67 DHFR variant will enable the design of more meaningful site-directed mutants where single substitutions per active site pore can be generated.
Department of Biochemistry, Cell and Molecular Biology, The University of Tennessee, Knoxville, Tennessee 37996-0840, USA. lzh@utk.edu
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Bradrick
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Shattuck
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Strader
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Wicker
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Eisenstein
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Howell
D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D005784:Gene Amplification; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000235:genetics*
D013762:Tetrahydrofolate Dehydrogenase