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

Spherulin 3a (S3a) from Physarum polycephalum represents the only known sin gle-domain member of the superfamily of beta gamma eye-lens crystallins. It shares the typical two Greek-key motif and is stabilized by dimerization a nd 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 reversibi lity without chemical modification of the protein during thermal denaturati on, 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 accord ing to N-2 --> 2 U, allowing thermodynamic parameters to be determined by L inear extrapolation to zero GdmCl concentration. The corresponding transiti on temperatures T-M, for the Ca2+-free and Ca2+-loaded protein were found t o 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 t o be determined. As a result, Delta C-p = 18 kJ/(K mol(dimer)) was calculat ed independent of Ca2+. No significant differences were obtained between th e free energy Delta G degrees calculated from Delta H-cal and T-M, and extr apolated 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 a t different temperatures for the Ca2+-free or the Ca2+-loaded protein, resp ectively. Within the limits of error, the Delta G degrees values extrapolat ed from the transitions of chemical denaturation to zero denaturant concent ration are identical with the calorimetric results. (C) 1999 Academic Press .