Thermodynamic analysis of the structural stability of phage 434 Cro protein

Thermodynamic parameters describing the phage 434 Cro protein have been det ermined 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 adeq uately described by the two-state model. The far-UV CD denaturation data yi eld 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 capaci ty change accompanying unfolding. Calorimetric data yield a temperature-ind ependent Delta C-p,u of 0.95 +/- 0.30 kcal mol(-1) K-1 or a temperaturl-dep endent 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 kno wn empirical correlations with structure. The free energy of unfolding is p H-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 o bserved 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 va lue reported for the putative monomeric lambda Cro, Since a protein's struc tural stability is important in determining its intracellular stability and turnover, the stability of Cro relative to the repressor could be a key co mponent of the regulatory circuit controlling the levels and, consequently, the functions of the two proteins in vivo.