Kinetic and thermodynamic stabilization of the betagamma-crystallin homolog spherulin 3a from Physarum polycephalum by calcium binding.
Globular proteins may be stabilized, either intrinsically, at the various levels of the structural hierarchy, or extrinsically, by ligand binding. In the case of the dormant all-beta protein spherulin 3a (S3a) from the slime mold Physarum polycephalum, binding of calcium ions causes extreme kinetic and thermodynamic stabilization. S3a is the only known single-domain member of the two Greek key superfamily of betagamma-crystallins sharing the extreme long-term stability of its homologs in vertebrate eye lens. Spectral analysis allows two Ca2+-binding sites with KD=9 microM and 200 microM to be distinguished. Unfolding in the absence and in the presence of Ca2+gives evidence for extreme kinetic stabilization of the protein: In the absence of Ca2+, the half-time of unfolding in 2. 5 M guanidinium chloride (GdmCl) equals 8.3 minutes, whereas in the presence of Ca2+, even in 7.5 M GdmCl, it exceeds nine hours. To reach the equilibrium of unfolding in the absence and in the presence of Ca2+takes one day and eight weeks, respectively. The corresponding Gibbs free energies (based on the two-state model) are 77 and 135 kJ/mol. Saturation of S3a with Ca2+leads to an upward shift of the temperature-induced equilibrium transition by ca 20 deg. C. The in situ Ca2+concentration in the spherules is sufficient for the complete complexation of S3a in vivo.
Kretschmar
M
M
Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Regensburg, D-93040, Germany.
Mayr
E M
EM
Jaenicke
R
R
D000818:Animals; D002118:Calcium / Q000737:chemistry*; D002942:Circular Dichroism; D003459:Crystallins / Q000737:chemistry*; D019791:Guanidine; D007700:Kinetics; D016839:Physarum polycephalum / Q000737:chemistry*; D011489:Protein Denaturation; D015800:Protozoan Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D003459:Crystallins; D015800:Protozoan Proteins; C108191:spherulin 3a protein, Physarum polycephalum; D019791:Guanidine; D002118:Calcium