A Folded Monomeric Intermediate in the Formation of Lambda Cro Dimer±DNA Complexes

Abstract

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.