Stability and kinetics of unfolding and refolding of cAMP receptor protein from Escherichia coli

Abstract

cAMP receptor protein (CRP) is involved in regulation of expression of several genes in Escherichia coli. The protein is a homodimer and each monomer is folded into two distinct structural domains. The mechanism of the biological activity of the protein may involve the interaction between the subunits and domains. In order to determine the interaction between the subunits or domains of CRP, we have studied the reversible denaturation of the protein by guanidine hydrochloride. The unfolding and refolding kinetics of CRP was monitored using stopped-flow fluorescence spectroscopy at 20 °C and pH 7.9. The results of CRP denaturation indicate that the transition can be described by a three-state model: (CRP(native))2 ⇆ 2 (CRP(native)) ⇆ 2 (CRP(denatured)). The faster process, characterized by the relaxation time τ2 = 80 ± 3 ms, corresponds to the dissociation of CRP dimer into monomers. The slower process has the relaxation time τ1 = 1.9 ± 0.1 s and corresponds to the cooperative unfolding of CRP monomer. The free energy change in the absence of denaturant upon CRP dissociation is Δ G(dis)°= 46.9 ± 2.5 kJ/mol and for monomer unfolding Δ G(unf)°= 30.9 ± 1.3 kJ/mol. The thermal unfolding of CRP was studied by circular dichroism and fluorescence spectroscopy at various guanidine hydrochloride concentrations. It has been found that the native protein is maximally stable at about 21 ± 0.3 °C and is denatured upon heating and cooling from this temperature. The apparent free energy change for CRP unfolding at 21°C is equal to 30.5 ± 0.4 kJ/ mol and the apparent specific heat change is equal to Δc(p,app) = 10.7 ± 0.7 kJ mol-1 K-1. The predicted values of cold denaturation midpoint is equal to t'(G) = -18.8 ± 1.5°C and for high-temperature transition t(G) = 63.1 ± 1.5 °C. The predicted midpoint of high-temperature unfolding transition is about the same as determined experimentally.