Thermodynamic analysis of the structural stability of phage 434 Cro protein

Thermodynamic parameters describing the phage 434 Cro protein have been determined by calorimetry and, independently, by far-UV circular dichroism CD measurements of isothermal urea denaturations and thermal denaturations at fixed urea concentrations. These equilibrium unfolding transitions are adequately described by the two-state model. The far-UV CD denaturation data yield average temperarure-independent values of 0.99 +/- 0.10 kcal mol(-1) M-1 for m and 0.98 +/- 0.05 kcal mol(-1) K-1 for Delta C-p,u, the heat capacity change accompanying unfolding. Calorimetric data yield a temperature-independent Delta C-p,u of 0.95 +/- 0.30 kcal mol(-1) K-1 or a temperaturl-dependent value of 1.00 +/- 0.10 kcal mol(-1) K-1 at 25 degrees C. Delta C-p,u and m determined for 434 Cro are in accord with values predicted using known empirical correlations with structure. The free energy of unfolding is pH-dependent, and the protein is completely unfolded at pH 2.0 and 25 OC as judged by calorimetry or CD. The stability of 434 Cro is lower than those observed for the structurally similar N-terminal domain of the repressor of phage 434 (R1-69) or of phage lambda (lambda(6-85)), but is close to the value reported for the putative monomeric lambda Cro, Since a protein's structural stability is important in determining its intracellular stability and turnover, the stability of Cro relative to the repressor could be a key component of the regulatory circuit controlling the levels and, consequently, the functions of the two proteins in vivo.