Thermodynamic analysis of unfolding and dissociation in lactose repressor protein

Lactose repressor protein, regulator of lac enzyme expression in Escherichi a coli, maintains its structure and function at extremely low protein conce ntrations (<10(-12) M). To examine the unfolding and dissociation of this t etrameric protein, structural transitions in the presence of varying concen trations of urea were monitored by fluorescence and circular dichroism spec troscopy, analytical ultracentrifugation, and functional activities. The sp ectroscopic data demonstrated a single cooperative transition with no evide nce of folded dimeric or monomeric species of this protein. These spectrosc opic transitions were reversible provided a long incubation step was employ ed in the refolding reaction at similar to 3 M urea. The refolded repressor protein possessed the same functional and structural properties as wild-ty pe repressor protein. The absence of concentration dependence expected for tetramer dissociation to unfolded monomer (M-4 <-> 4U) in the spectral tran sitions indicates that the disruption of the monomer-monomer interface and monomer unfolding are a concerted reaction (M-4 <->U-4) that may occur prio r to the dissociation of the dimer-dimer interface. Thus, we propose that t he unfolded monomers remain associated at the C-terminus by the 4-helical c oiled-coil structure that forms the dimer-dimer interface and that this int ermediate is the end point detected in the spectral transitions. Efforts to confirm the existence of this species by ultracentrifugation were inhibite d by the aggregation of this intermediate. Based upon these observations, t he wild-type fluorescence and CD data were fit to a model, M-4 <-> U-4, whi ch resulted in an overall Delta G degrees for unfolding of 40 kcal/mol. Usi ng a mutant protein, K84L, in which the monomer-monomer interface is stabil ized, sedimentation equilibrium results demonstrated that the dimer-dimer i nterface of inc repressor could persist at higher levels of urea than the m onomer-monomer interface. The tetramer-dimer transition monitored using thi s mutant repressor yields a Delta G degrees of 20.4 kcal/mol. Using this fr ee energy value for the dissociation process of U-4 <-> 4U, an overall free energy change of similar to 60 kcal/mol was calculated for dissociation of all interfaces and unfolding of the tetrameric lac repressor, reflecting t he exceptional stability of this protein.