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A folded monomeric intermediate in the formation of lambda Cro dimer-DNA complexes

R Jana1, T R Hazbun, A K Mollah

  • 1Department of Biological Sciences, University of Notre Dame, Notre Dame, IN, 46556, USA.

Journal of Molecular Biology
|November 5, 1997
PubMed
Summary

The bacteriophage lambda Cro repressor exists as a folded monomer at low concentrations, not a dimer, influencing its DNA binding. Engineered variants reveal residue 58 stabilizes this monomeric form.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Structural Biology

Background:

  • The bacteriophage lambda Cro repressor regulates viral gene expression through DNA binding.
  • Understanding the oligomeric state and folding of Cro repressor is crucial for its function.

Purpose of the Study:

  • To characterize the folding, dimerization, and DNA binding equilibria of the Cro repressor.
  • To investigate the role of specific residues in protein stability and DNA binding.

Main Methods:

  • Electrophoretic mobility shift assays (EMSA) to study DNA binding.
  • Sedimentation equilibrium and gel filtration chromatography to determine dissociation constants.
  • Guanidine hydrochloride (GdnHCl) denaturation to assess protein stability.

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Main Results:

  • A folded monomeric species of Cro repressor is significantly populated, even under conditions favoring dimer formation.
  • DNA binding isotherms indicate monomeric species predominate at nanomolar concentrations.
  • Engineered variants, particularly Cro F58W, show increased stability of the folded monomer.

Conclusions:

  • The predominant free Cro repressor species at functional concentrations is monomeric and folded.
  • Residue 58 plays a dual role in stabilizing both the monomeric intermediate and the dimer interface.
  • These findings refine our understanding of Cro repressor's mechanism of action and protein folding principles.