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-do cumented phenomenon, little is known about the molecular details of th e process, In this study, the parameters describing the denaturation t hermodynamics of residues 6-85 of the N-terminal domain of lambda repr essor have been determined by fitting the three-dimensional thermal-ur ea denaturation surface obtained by circular dichroism. The shape of t he surface shows cold denaturation at low temperatures and urea concen trations above 2 M, which allows accurate determination of the apparen t heat capacity of denaturation (Delta C-p). Denaturation curves based on aromatic H-1 NMR spectra give identical denaturation curves, confi rming purely two-state folding under all conditions studied. The denat uration surface can be fit with constant Delta C-p and delta In K-D/de lta[urea] (K-D is the equilibrium constant for denaturation), consiste nt with a thermodynamically invariant denatured state. In addition, th e 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 co ld and heat denatured states, in the presence of 3 M urea, are thermod ynamically and conformationally equivalent.