| Čoahkkáigeassu: | The folding, dimerization and DNA binding equilibria of the bacterio-
phage lambda Cro repressor have been characterized. Comparison with
four engineered variants shows that a folded monomeric species is sub-
stantially populated under conditions used for the formation of dimer±
DNA complexes. Although Cro dimers are the only DNA-bound species
observed in electrophoretic mobility shift assays, cooperativity in Cro ±
DNA binding isotherms shows that the predominant free protein species
is monomeric at nanomolar concentrations. Micromolar dissociation con-
stants for Cro dimers have been measured in the absence of DNA by
sedimentation equilibrium and gel ®ltration chromatography. Denatura-
tion of Cro dimers in the 10 to 100 micromolar concentration range by
guanidine hydrochloride (GdnHCl) is well modeled as a two-state pro-
cess, with folded dimers and unfolded monomers as the only signi®-
cantly populated species. However, linear extrapolation of this composite
unfolding and dimer dissociation free energy predicts a nanomolar dis-
sociation constant in the absence of denaturant. This extrapolation is
clearly inconsistent with the DNA binding and hydrodynamic measure-
ments. Our interpretation of these results is that the monomeric species
detected in DNA binding and hydrodynamic experiments is predomi-
nantly folded. The stability of the folded monomeric species can be calcu-
lated as the difference between the dimerization free energy determined
from hydrodynamic measurements and the folding free energy extrapo-
lated from GdnHCl denaturation. The calculated stability of the Cro
F58W monomer is greater than that of the wild-type Cro monomer.
Thus, residue 58, which makes critical intermolecular contacts across the
dimer interface, is also involved in intramolecular stabilization of the
monomeric intermediate.
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