Rational Design and Analysis of Calcium Binding Protein

描述：(申请人提供)本项目的目标是设计和 分析钙结合蛋白以了解关键部位 控制钙结合亲和力的因素，如配体类型和电荷。 我们的长期目标是了解钙调节的机制。 信号和细胞黏附。由于同位素标记的金属离子可以通过 放射、核磁共振或化学手段，我们在设计金属装订方面的成功 进入任意蛋白质的位点可能会导致开发有用的新方法 用于诊断测试和化疗的试剂。 我们假设每个残基作为配基使用的频率在已知 钙结合部位代表其相对的钙结合能力。两者都有 带负电荷的残基与羧基的侧链刚性 确定钙亲和力的顺序：天冬氨酸、丝氨酸、苏氨酸、谷氨酸氨基转移酶。我们的 新的方法使我们能够最大限度地减少整体蛋白质对全球的影响 仅用少量残基保持蛋白质结构不变的构象 变化。在这种情况下，由于维持了蛋白质环境，所以测量到的 钙结合亲和力可直接与局部结构相关 金属结合部位的特征。残余型的贡献 (旋转异构体)，配体位置上的电荷分布，以及与 亲和力将被测量。帮助我们设计蛋白质中的钙结合部位 并预测其与钙结合的亲和力，我们还建议解剖 引入电荷对钙亲和力的贡献 具有不同静电排列的钙中心周围的残留物。我们 进一步建议表征Ca(II)结合部位(配体类型和 几何)使用高分辨率方法。钙离子结合的比较 用高分辨率方法确定的蛋白质中的位点与 将执行最初设计的目标，以优化我们的方法 钙的设计

DESCRIPTION: (provided by applicant) The goal of this project is to design and analyze calcium-binding proteins in order to gain an understanding of key site factors, such as ligand type and charge, that control calcium-binding affinity. Our long-term goal is to understand the mechanism of calcium-modulated signaling and cell adhesion. Since isotope-labeled metal ions can be tracked by radiological, NMR or chemical means, our success in designing metal binding Sites into arbitrary proteins will likely lead to new ways of developing useful reagents for diagnostic tests and chemotherapy. We hypothesize that the frequency of each residue used as a ligand in known calcium-binding sites represents its relative calcium binding ability. Both negatively charged residues and the side chain rigidity of the carboxyl group determine the order for calcium affinity: Asp> Glu> Asn, Ser, Thr> Gln. Our novel approach allows us to minimize global effects from the overall protein conformation by keeping the same protein structure with only a few residues changed. In this case since protein environment is maintained, the measured calcium binding affinity can be directly correlated to the local structural features of the metal-binding sites. The contribution of the residue type (rotamer), charge distribution at the ligand positions, and bond length to the affinity will be measured. To help us design calcium-binding sites in proteins and predict their calcium-binding affinity, we also propose to dissect the contribution of charge distribution to calcium affinity by introducing charged residues around the calcium sites with different electrostatic arrangements. We further propose to characterize the Ca(II) binding sites (ligand types and the geometry) using high resolution methods. A comparison of the calcium binding sites in proteins determined by high-resolution methods with those of the originally designed target will be carried out to optimize our methods for design of calcium