Oligomerization state of S100B at nanomolar concentration determined by large-zone analytical gel filtration chromatography
Oligomerization state of S100B at nanomolar concentration determined by large-zone analytical gel filtration chromatography
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DOI：
10.1002/pro.5560060721
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发表时间：
1997-07-01
期刊：
影响因子：
8
通讯作者：
Weber, DJ
中科院分区：
文献类型：
--
作者：
Drohat, AC;Nenortas, E;Weber, DJ
S100B是一种钙离子结合蛋白，是一种已知的非共价结合二聚体S100B(PP)，在还原条件下，浓度较高(0.2-3.0 mM)。Apo-S100B(PP)的溶液结构表明，亚基以反平行的方式结合，在二聚体界面形成一个紧密堆积的疏水核心，涉及八个螺旋中的六个和C-末端环(Drohat AC、Amburgey JC、Abildgaard F、Starich MR、Balessi D、Weber DJ)。1996年。核磁共振波谱测定大鼠apo-S100B(PP)的溶液结构生物化学35：11577-11588)。然而，已知的C-末端环也参与了S100B与靶蛋白的结合，因此它参与二聚体界面的作用引发了关于二聚体S100B(β)的生理相关性的问题。因此，我们用S-35标记的S100B进行了大面积分析凝胶过滤层析，研究了S100B在低浓度(1-10,000 nm)下的齐聚状态。我们发现S100B以非共价结合的二聚体S100B(PP)的形式存在(>；99%)，在存在或不存在饱和水平的钙离子的情况下，S100B(PP)的亚基浓度为1 nM(500 pm二聚体)，这意味着解离常数在皮摩尔范围或更低。这些结果首次表明，在还原环境和生理浓度下，S100B在存在或不存在钙离子的情况下以二聚体S100B(β)的形式存在，并且非共价二聚体很可能是S100B呈现给靶蛋白的形式。
S100B is a Ca2+-binding protein known to be a noncovalently associated dimer, S100B(PP), at high concentrations (0.2-3.0 mM) under reducing conditions. The solution structure of apo-S100B(PP) 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(PP) 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 S-35-labeled S100B. We found that S100B exists (>99%) as a non-covalently associated dimer, S100B(PP), 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.