Rational Design and Analysis of Calcium Binding Protein

钙结合蛋白的合理设计与分析

• 批准号: 6769888
• 负责人: Jenny J. Yang
• 金额: $ 21.09万
• 依托单位: GEORGIA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6769888
• 关键词: CD2 molecule; X ray crystallography; binding sites; calcium binding protein; circular dichroism; conformation; fluorescent dye /probe; gel filtration chromatography; ionic bond; ligands; mass spectrometry; mathematical model; molecular dynamics; molecular shape; nuclear magnetic resonance spectroscopy; nucleic acid sequence; peptide chemical synthesis; protein engineering; protein structure; site directed mutagenesis; stable isotope double label

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

描述：（由申请人提供）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，例如配体类型和电荷。 我们的长期目标是了解钙调节 信号传导和细胞粘附。由于同位素标记的金属离子可以通过 放射性、核磁共振或化学手段，我们在设计金属结合物方面的成功 任意蛋白质的位点将可能导致开发有用的 用于诊断测试和化疗的试剂。 我们假设，在已知的细胞中用作配体的每个残基的频率 钙结合位点代表其相对钙结合能力。两 带负电荷的残基和羧基的侧链刚性 确定钙离子亲和力顺序为：Asp> Glu> Asn，Ser，Thr> Gln。我们 一种新的方法使我们能够最大限度地减少整体蛋白质的全局影响， 通过保持相同的蛋白质结构，只有几个残基， 变了在这种情况下，由于维持了蛋白质环境，因此测量的 钙结合亲和力可以直接与局部结构相关， 金属结合位点的特征。剩余类型的贡献 （旋转异构体）、配体位置处的电荷分布以及与配体的键长。 将测量亲和力。帮助我们设计蛋白质中的钙结合位点 并预测其钙结合亲和力，我们还建议解剖 电荷分布对钙亲和力的贡献 不同静电排列的钙位点周围的残基。我们 进一步提出表征Ca（II）结合位点（配体类型和 几何学）使用高分辨率方法。钙结合的比较 通过高分辨率方法确定的蛋白质中的位点与 我们将按照最初设计的目标来优化我们的方法， 钙的设计