Kretschmar, Michael and Jaenicke, Rainer (1999) Stability of a homo-dimeric Ca2+-binding member of the beta gamma-crystallin superfamily: DSC measurements on spherulin 3a from Physarum polycephalum. JOURNAL OF MOLECULAR BIOLOGY, 291 (5). pp. 1147-1153. ISSN 0022-2836, 1089-8638
Full text not available from this repository. (Request a copy)Abstract
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.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | DOMAIN INTERACTIONS; B-CRYSTALLIN; EYE LENS; TEMPERATURE; DEPENDENCE; PROTEINS; WATER; PH; beta gamma-crystallins; calcium binding; differential scanning calorimetry; dimer; spherulin |
| Subjects: | 500 Science > 570 Life sciences |
| Divisions: | Biology, Preclinical Medicine > Institut für Biophysik und physikalische Biochemie |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 21 Dec 2022 08:31 |
| Last Modified: | 21 Dec 2022 08:31 |
| URI: | https://pred.uni-regensburg.de/id/eprint/49004 |
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