STRUCTURE/FUNCTION OF S100 PROTEIN

在过去的三年中，我们(I)准备了过表达载体 产生高产量(大于30毫克/升)的S100B(贝塔)， S100A1和S100L，(Ii)确定了apo-和 利用异核以高分辨率结合Ca2+的S100B(Beta) 多维核磁共振波谱，(III)收集液体中的核磁共振数据 测量偶极耦合值所需的结晶介质，(Iv) 表明二聚体S100B(Beta)是与生理相关的 该蛋白的齐聚状态，以及(V)确定了S100B (β)通过蛋白激酶C抑制P53的磷酸化 (PKC)以一种钙依赖的方式，这种抑制是结果 S100B(Beta Beta)直接与C-末端调控相互作用 P53的结构域。 我们计划继续表征钙离子依赖的相互作用 S100B(Beta)与目的蛋白结合。首先，我们将完善我们的 载脂蛋白和钙离子负载的S100B(β-β)的核磁共振结构 偶极耦合约束。我们还将确定3D结构 钙离子结合的S100B(β-β)与来源于 P53的C-末端调节域(残基367-388)。这将是 代表S100-靶蛋白复合体的第一个3D结构。这个 我们还将研究锌离子与S100B(β-β)的结合。 将确定锌离子结合部位是否与靶重叠 蛋白质站点。计划对所有人进行异核弛豫测量 我们为了阐明钙离子和靶向 蛋白质结合影响S100B(βETA)的动态过程。最后， 将确定S100A和S100L的三维溶液结构并 与S100B(测试版)相比。这样做的目标是 鉴定这两种蛋白质的结构特性 由于它们对钙离子具有较高的亲和力和靶标的特异性 蛋白质结合。 我们的目标是鉴定S100-靶蛋白 与抑制它们的长期目标相互作用。因此， 结构研究，结合定点突变， 热力学约束和动态测量将用于 在原子分辨率下，描述特定的 S100B(β-β)与不同蛋白质靶标的残留量 解决方案。基于这一信息的抑制剂可能与 治疗癌症和癌症等疾病中发现的不受控制的细胞生长 阿尔茨海默氏症。

During the last 3 years we (i) prepared overexpression vectors that produce high yields (greater than 30 mg/liter) of S100B (beta beta), S100A1, and S100L, (ii) determined the solution structures of apo- and Ca2+-bound S100B (beta beta) at high resolution using heteronuclear multidimensional NMR spectroscopy, (iii) collected NMR data in liquid crystalline media necessary to measure dipolar coupling values, (iv) showed that dimeric S100B (beta beta) is the physiologically relevant oligomerization state of this protein, and (v) determined that S100B (beta beta) inhibits p53 phosphorylation by protein kinase C kinase C (PKC) in a Ca2+-dependent manner, and that this inhibition is the result of S100B (beta beta) interacting directly with the C-terminal regulatory domain of p53. We plan to continue characterizing the Ca2+-dependent interaction of S100B (beta beta) with target proteins. First, we will refine our previous NMR structures of apo- and Ca2+-loaded S100B (beta beta) using dipolar coupling constraints. We will also determine the 3D structure of Ca2+-bound S100B (beta beta) complexed with peptide derived from the C-terminal regulatory domain of p53 (residues 367-388). This will represent the first 3D structure of a S100-target protein complex. The binding of Zn2+ to S100B (beta beta) will also be characterized, and we will determine whether the Zn2+ binding site overlaps with the target protein site. Heteronuclear relaxation measurements are planned for all of the structures that we solve in order to clarify how Ca2+ and target protein binding affects dynamic processes in S100B (beta eta). Lastly, the 3D solution structures of S100A and S100L will be determined and compared to S100B (beta beta). This will be done with the goal of identifying the structural properties of these two proteins that lead to their higher affinity for Ca2+ and their specificity in target protein binding. Our effort is directed towards characterizing S100-target protein interactions with the long-range goal of inhibiting them. Therefore, structural studies, together with site-directed mutagenesis, thermodynamic binding, and dynamic measurements will be used to characterize, at atomic resolution, the interaction between specific residues of S100B (beta beta) with those of various protein targets in solution. An inhibitor based on this information could be relevant to treating uncontrolled cell growth found in diseases such as cancer and Alzheimer's disease.