Helium Recovery System for CCRC NMR Facility

CCRC NMR 设施的氦气回收系统

• 批准号: 9987915
• 负责人: JAMES H. PRESTEGARD
• 金额: $ 16.73万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-07-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9987915
• 关键词: Adhesions; Affect; Biological; CEACAM1; CEACAM5 gene; Carbohydrates; Cell Adhesion; Cell Communication; Cell surface; Cells; Complex; Cytoplasmic Tail; Development; Dimerization; Disease; Elements; Engineering; Environment; Family; Funding; Genes; Health; Helium; Heterogeneity; Human; Human body; Immunoglobulins; Inflammatory; Invaded; Isotope Labeling; Label; Laboratories; Malignant Neoplasms; Mammalian Cell; Marshal; Mass Spectrum Analysis; Mediating; Membrane; Methodology; Methods; Molecular Conformation; Organism; Polysaccharides; Production; Proteins; Recovery; Research; Research Personnel; Role; Sampling; Signal Transduction; Signaling Molecule; Signaling Protein; Structure; System; Tissues; Work; base; carcinoembryonic antigen-related cell adhesion molecules; dimer; extracellular; glycosylation; human disease; improved; member; novel; novel diagnostics; parent grant; pathogen; screening

RESEARCH STRATEGY Abstract of parent grant: GM033225 Mammalian cells must respond to signals in their environment in complex ways in order to properly develop, form tissues, and marshal a defense against invading organisms. Much of the cell-cell communication needed is provided by a class of cell adhesion and signaling proteins that share a topology that includes an extracellular variable immunoglobulin-like (IgV) domain, one or more constant immunoglobulin-like (IgC) domains, a membrane anchor, and sometimes a cytoplasmic domain that carries signaling elements. The extra cellular domains of these molecules are heavily glycosylated and the glycans (carbohydrates) on these domains mediate or modify interactions with similar molecules on other cells and thus modulate biologically important signals. CEACAMs (carcinoembryonic antigen related cell adhesion molecules) are members of this class. Our work in the previous funding segment focused on CEACAM1, the product of one of twelve genes encoding CEACAMs in humans. We were able to produce a dimer structure of the IgV domain of CEACAM1, but more importantly, show that dimerization was affected by even minimal glycosylation. There are relatively few systematic structural studies of the effects of glycosylation in the CEACAMs or any of the related cell- surface signaling molecules, despite clear evidence that glycosylation modulates function. This arises because of the heterogeneity of native glycosylation, the challenges in preparing samples that are homogeneously glycosylated, and the limitations glycosylation places on common structure determination methods. Hence, to undertake this study we have assembled a team of investigators from three laboratories with complementary expertise in mammalian cell expression and glycan engineering, mass spectrometry-based protein and carbohydrate analysis, and NMR-based structure determination of glycosylated proteins. Aims include the production of specific glycoforms of IgV-IgC constructs of CEACAM1 and CEACAM5 with isotopic labeling suitable for NMR structure determination, mass spectrometry based screening of glycoforms to identify those displaying altered conformation or dimerization tendencies, and NMR determination of dimer structures and glycan interactions of targeted glycoforms using novel sparse-label methodology. Understanding how glycan interactions can alter structure and modulate signaling in the CEACAMs can impact directly on human health, but more importantly, the methods developed through work on CEACAMs can have a broader impact by allowing research on the many other glycosylated proteins found in the human body.

研究策略 父级赠款摘要：GM033225 哺乳动物细胞必须以复杂的方式响应其环境中的信号才能正确 开发，形成组织和元帅，防御入侵生物。大部分细胞细胞 所需的交流是由一类细胞粘附和信号蛋白提供的，它们共享拓扑， 包括细胞外变量的免疫球蛋白样（IGV）结构域，一个或多个恒定的免疫球蛋白样 （IGC）结构域，膜锚，有时是带有信号元件的细胞质结构域。这 这些分子的额外细胞结构域是大量的糖基化，糖基（碳水化合物）在这些上是 域介导或修饰与其他细胞上相似分子的相互作用，因此在生物学上调节 重要信号。 CECAMS（Carcinoembryonic抗原相关的细胞粘附分子）是其中的成员 班级。我们在以前的资金部分的工作重点是CEACAM1，这是十二个基因之一的产物 编码人类中的CECAM。我们能够产生CeCAM1 IgV结构域的二聚体结构， 但更重要的是，表明二聚化受到最小糖基化的影响。有相对的 很少有系统的结构研究对CECAM或任何相关细胞中糖基化的影响 尽管有明确的证据表明糖基化调节了功能，但表面信号分子。这是因为 在天然糖基化的异质性中，制备同质样品的挑战 糖基化，限制糖基化位置在共同的结构测定方法上。因此，要 进行这项研究，我们组建了来自三个实验室的调查员团队 哺乳动物细胞表达和聚糖工程的专业知识，基于质谱的蛋白质和 碳水化合物分析和基于NMR的糖基化蛋白的结构测定。目标包括 具有同位素标记的CECAM1和CECAM5的IGV-IGC构建体的特定糖基型 适用于NMR结构确定，基于质谱的糖型筛选，以识别这些 显示改变的构象或二聚化趋势，以及二聚体结构的NMR确定和 使用新型的稀疏标签方法论，靶向糖型的聚糖相互作用。了解聚糖 相互作用可以改变结构，并调节CEACAM中的信号传导会直接影响人类健康， 但更重要的是，通过在CEACAMS上开发的方法可以通过 允许研究人体中发现的许多其他糖基化蛋白质。