https://repository.auw.edu.bd/handle/123456789/829 Tue, 14 Jul 2026 16:53:59 GMT 2026-07-14T16:53:59Z https://repository.auw.edu.bd/handle/123456789/1033 A Folded Monomeric Intermediate in the Formation of Lambda Cro Dimer±DNA Complexes . Mollah, A. K. M. M 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. Wed, 01 Jan 1997 00:00:00 GMT https://repository.auw.edu.bd/handle/123456789/1033 1997-01-01T00:00:00Z