Heat and cold denatured states of monomeric lambda repressor are thermodynamically and conformationally equivalent

Although the denaturation of proteins by low temperatures is a well-documented phenomenon, little is known about the molecular details of the process, In this study, the parameters describing the denaturation thermodynamics of residues 6-85 of the N-terminal domain of lambda repressor have been determined by fitting the three-dimensional thermal-urea denaturation surface obtained by circular dichroism. The shape of the surface shows cold denaturation at low temperatures and urea concentrations above 2 M, which allows accurate determination of the apparent heat capacity of denaturation (Delta C-p). Denaturation curves based on aromatic H-1 NMR spectra give identical denaturation curves, confirming purely two-state folding under all conditions studied. The denaturation surface can be fit with constant Delta C-p and delta In K-D/delta[urea] (K-D is the equilibrium constant for denaturation), consistent with a thermodynamically invariant denatured state. In addition, the aromatic H-1 NMR spectrum of the cold denatured state at 0 degrees C in 3 M urea is essentially identical to the spectrum at 70 degrees C in 3 M urea. These observations indicate that the structures of the cold and heat denatured states, in the presence of 3 M urea, are thermodynamically and conformationally equivalent.