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Related Experiment Videos

Effects of single-tryptophan mutations on R67 dihydrofolate reductase.

F W West1, H S Seo, T D Bradrick

  • 1Department of Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville, 37996-0840, USA.

Biochemistry
|March 29, 2000
PubMed
Summary

Mutations in R67 dihydrofolate reductase (DHFR) reveal key structural roles of tryptophan residues. W45F mutations maintain enzyme activity, while W38F mutations destabilize the tetramer, impacting trimethoprim resistance.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Structural Biology

Background:

  • R67 dihydrofolate reductase (DHFR) is an R-plasmid-encoded enzyme conferring resistance to trimethoprim.
  • This enzyme is structurally distinct from chromosomal DHFRs and functions as a homotetramer with D(2) symmetry.

Purpose of the Study:

  • To investigate the structural and functional roles of specific tryptophan residues (W38 and W45) in R67 DHFR.
  • To determine the impact of mutations at these residues on enzyme activity, stability, and quaternary structure.

Main Methods:

  • Construction and characterization of single-tryptophan mutant genes (W45F and W38F).
  • Enzyme activity assays and fluorescence spectroscopy to monitor ligand binding and conformational changes.
  • Equilibrium unfolding/folding studies to assess protein stability.

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Main Results:

  • The W45F mutant retained full enzyme activity, with W38 fluorescence effectively reporting on ligand binding and pH-dependent dimer-ligand equilibrium.
  • Simultaneous W38F mutations destabilized the tetramer, leading to an inactive dimeric form, similar to dimeric wild-type R67 DHFR.
  • Fluorescence emission spectra indicated W38 contributes significantly to the signal of wild-type R67 DHFR.

Conclusions:

  • Tryptophan residues W38 and W45 play distinct roles in the structural integrity and function of R67 DHFR.
  • W45 is crucial for maintaining enzyme activity and monitoring ligand interactions, while W38 is critical for tetramer stability.