Role of the N-terminal helix I for dimerization and stability of the calcium-binding protein S100B.
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.
Ferguson
Peter L
PL
Department of Biochemistry and R. S. McLaughlin Macromolecular Structure Facility, The University of Western Ontario, London, Ontario N6A 5C1, Canada.
Shaw
Gary S
GS
D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D017931:DNA Primers; D019281:Dimerization; D006801:Humans; D008958:Models, Molecular; D016296:Mutagenesis; D009414:Nerve Growth Factors / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; D013816:Thermodynamics
D002135:Calcium-Binding Proteins; D017931:DNA Primers; D009414:Nerve Growth Factors; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; C579160:S100B protein, human