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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
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.
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.
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.