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 Ca(2+)-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 N2-->2 U, allowing thermodynamic parameters to be determined by linear extrapolation to zero GdmCl concentration. The corresponding transition temperatures TM for the Ca(2+)-free and Ca(2+)-loaded protein were found to be 65 and 85 degrees C, the enthalpy changes delta Hcal, 800 and 1280 kJ/mol(dimer), respectively. The strong dependencies of TM and delta Hcal on the GdmCl concentration allow the molar heat capacity change delta Cp to be determined. As a result, delta Cp = 18 kJ/(K mol(dimer)) was calculated independent of Ca2+. No significant differences were obtained between the free energy delta G degree calculated from delta Hcal and TM, 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 Ca(2+)-free or the Ca(2+)-loaded protein, respectively. Within the limits of error, the delta G degree values extrapolated from the transitions of chemical denaturation to zero denaturant concentration are identical with the calorimetric results. Study holds ProTherm entries: 6190, 6191, 6192, 6193, 6194, 6195, 6196, 6197, 6198, 6199, 6200, 6201, 6202 Extra Details: additive : EDTA(1 mM), beta gamma-crystallins; calcium binding;,differential scanning calorimetry; dimer; spherulin
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:31 p.m.
|Number of data points||39|
|Proteins||Spherulin-3A ; Spherulin-3A|
|Assays/Quantities/Protocols||Experimental Assay: dHcal ionic:CaCl2: 3 mM, details:Additives ; Experimental Assay: Tm ionic:CaCl2: 3 mM, details:Additives ; Experimental Assay: dHvH ionic:CaCl2: 3 mM, details:Additives ; Experimental Assay: dHcal ionic:: , details:Additives EDTA (1 mM), ; Experimental Assay: Tm ionic:: , details:Additives EDTA (1 mM), ; Experimental Assay: dHvH ionic:: , details:Additives EDTA (1 mM),|
|Libraries||Mutations for sequence MSVCKGVSGNPAKGEVFLYKHVNFQGDSWKVTGNVYDFRSVSGLNDVVSSVKVGPNTKAFIFKDDRFNGNFIRLEESSQVTDLTTRNLNDAISSIIVATFESA|