Stability of a homo-dimeric Ca2+-binding member of the βγ-crystallin superfamily:: DSC measurements on spherulin 3a from Physarum polycephalum

Spherulin 3a (S3a) from Physarum polycephalum represents the only known single-domain member of the superfamily of beta gamma eye-lens crystallins. It shares the typical two Greek-key motif and is stabilized by dimerization and Ca2+-binding. The temperature and denaturant-induced unfolding of S3a in the absence and in the presence of Ca2+ were investigated by differential scanning calorimetry and fluorescence spectroscopy. To accomplish reversibility without chemical modification of the protein during thermal denaturation, the only cysteine residue (Cys4) was substituted by serine; apart from that, the protein was destabilized by adding 0.5-1.8 M guanidinium chloride (GdmCl). The Cys4Ser mutant was found to be indistinguishable from natural S3a. The equilibrium unfolding transitions obey the two-state model according to N-2 --> 2 U, allowing thermodynamic parameters to be determined by Linear extrapolation to zero GdmCl concentration. The corresponding transition temperatures T-M, for the Ca2+-free and Ca2+-loaded protein were found to be 65 and 85 degrees C, the enthalpy changes Delta H-cal, 800 and 1280 kJ/mol(dimer), respectively. The strong dependencies of T-M, and Delta H-cal on the GdmCl concentration allow the molar heat capacity change Delta C-p to be determined. As a result, Delta C-p = 18 kJ/(K mol(dimer)) was calculated independent of Ca2+. No significant differences were obtained between the free energy Delta G degrees calculated from Delta H-cal and T-M, and extrapolated from the stability curves in the presence of different amounts of denaturant. The free energy derived from thermal unfolding was confirmed by the spectral results obtained from GdmCl-induced equilibrium transitions at different temperatures for the Ca2+-free or the Ca2+-loaded protein, respectively. Within the limits of error, the Delta G degrees values extrapolated from the transitions of chemical denaturation to zero denaturant concentration are identical with the calorimetric results. (C) 1999 Academic Press.