Human S100B(beta beta) is a small intracellular EF-hand calcium-binding protein that consists of two noncovalently associated 91-residue beta monomers. The three-dimensional structures of S100B reveal the dimer interface consists of four alpha-helices (I, I' and IV, IV') packed in an X-type bundle. In this study, guanidine hydrochloride denaturation and dynamic light scattering were used to assess the impact of single (L3A, L3S, M7A, I11A, F14A) and double (L3A/I11A and L3A/F14A) substitution mutations in helix I on the stability and dimerization propensity of S100B. The free energy of unfolding (Delta G(u)) of wild-type apo-S100B was determined to be 72.4 +/- 4.0 kJ mol(-1), consistent with it being the most stable calcium-binding protein to date. The order of stability of the mutants in their apo form is S100B > L3A > L3S > I11A > M7A approximately L3A/I11A > F14A > L3A/F14A. Further, there is a strong correlation between the stability and the cooperativity of unfolding. Each mutation proved to be more stable in its calcium form compared to its apo form. The calcium-bound L3S substitution proved to be significantly more stable than calcium-saturated S100B, whereas the L3A, I11A, and L3A/I11A mutants are only slightly more stable than the wild-type protein. The F14A and L3A/F14A mutants are significantly reduced in stability, even in the presence of calcium. Study holds ProTherm entries: 13045, 13046, 13047, 13048, 13049, 13050, 13051, 13052, 13053, 13054, 13055, 13056, 13057, 13058, 13059, 13060 Extra Details: apo form; 1mM DTT and 1mM EDTA were added in the experiment. EF-hand; free energy; cooperativity; calcium
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:44 p.m.
|Number of data points||54|
|Proteins||Protein S100-B ; Protein S100-B|
|Assays/Quantities/Protocols||Experimental Assay: Cm prot_conc:2.2 to 26 mM ; Experimental Assay: m prot_conc:2.2 to 26 mM ; Experimental Assay: dG_H2O prot_conc:2.2 to 26 mM ; Experimental Assay: ddG_H2O ; Experimental Assay: Cm prot_conc:2.2 to 26 micro M ; Experimental Assay: m prot_conc:2.2 to 26 micro M ; Experimental Assay: dG_H2O prot_conc:2.2 to 26 micro M|
|Libraries||Mutations for sequence SELEKAMVALIDVFHQYSGREGDKHKLKKSELKELINNELSHFLEEIKEQEVVDKVMETLDNDGDGECDFQEFMAFVAMVTTACHEFFEHE|