Oligomerization state of S100B at nanomolar concentration determined by large-zone analytical gel filtration chromatography.
S100B is a Ca(2+)-binding protein known to be a non-covalently associated dimer, S100B(beta beta), at high concentrations (0.2-3.0 mM) under reducing conditions. The solution structure of apo-S100B (beta beta) shows that the subunits associate in an antiparallel manner to form a tightly packed hydrophobic core at the dimer interface involving six of eight helices and the C-terminal loop (Drohat AC, Amburgey JC, Abildgaard F, Starich MR, Baldisseri D, Weber DJ. 1996. Solution structure of rat apo-S100B (beta beta) as determined by NMR spectroscopy. Biochemistry 35:11577-11588). The C-terminal loop, however, is also known to participate in the binding of S100B to target proteins, so its participation in the dimer interface raises questions as to the physiological relevance of dimeric S100B (beta beta). Therefore, we investigated the oligomerization state of S100B at low concentrations (1-10,000 nM) using large-zone analytical gel filtration chromatography with 35S-labeled S100B. We found that S100B exists (> 99%) as a non-covalently associated dimer, S100B (beta beta), at 1 nM subunit concentration (500 pM dimer) in the presence or absence of saturating levels of Ca2+, which implies a dissociation constant in the picomolar range or lower. These results demonstrate for the first time that in reducing environments and at physiological concentrations, S100B exists as dimeric S100B (beta beta) in the presence or absence of Ca2+, and that the non-covalent dimer is most likely the form of S100B presented to target proteins.
Drohat
A C
AC
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore 21201, USA.
Nenortas
E
E
Beckett
D
D
Weber
D J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000737:chemistry* / Q000187:drug effects; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000187:drug effects; D002850:Chromatography, Gel / Q000379:methods; D019281:Dimerization; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000737:chemistry* / Q000187:drug effects; D011487:Protein Conformation; D051381:Rats; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; D017386:Sequence Homology, Amino Acid; D012996:Solutions
D001059:Apoproteins; D002135:Calcium-Binding Proteins; D009414:Nerve Growth Factors; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; C579158:S100b protein, rat; D012996:Solutions; D002118:Calcium