NEW NMR METHODOLOGY FOR CHARACTERIZING CARBOHYDRATE-PROTEIN INTERACTIONS

表征碳水化合物-蛋白质相互作用的新核磁共振方法

• 批准号: 8168839
• 负责人: JAMES H. PRESTEGARD
• 金额: $ 10.11万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-10 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8168839
• 关键词: Amides; B-Cell Activation; Carbohydrates; Cell Aggregation; Cell Differentiation process; Cells; Computer Retrieval of Information on Scientific Projects Database; Data; Disease; Environment; Fucosyltransferase; Funding; Glycoproteins; Grant; Inflammation; Institution; Label; Ligand Binding; Ligands; Malignant Neoplasms; Methodology; Modeling; Nuclear Magnetic Resonance; Oligosaccharides; Polysaccharides; Process; Protein-Carbohydrate Interaction; Proteins; Relaxation; Research; Research Personnel; Residual state; Resources; Retrieval; ST6Gal I; Sialic Acids; Sialyltransferases; Signal Transduction; Site; Source; Structural Models; Transferase; United States National Institutes of Health; Virus Diseases; Work; analog; drug development; protein complex; research study; tool; vector

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The formation of carbohydrate-protein complexes is important in a variety of processes involving the interaction of a cell with its environment. These processes include natural ones such as cell differentiation, cell aggregation, and cell signaling; they also include disease processes such as viral infection, malignancy, and unwanted inflammation. The development of drugs to moderate natural processes and inhibit disease processes begins with accurate structural models for oligosaccharide-protein interactions. These models have been difficult to get by traditional Nuclear Magnetic Resonance (NMR) approaches because of the dearth of short-distance NOE contacts in oligosaccharide-protein complexes. We are developing a number of experiments and analysis tools that allow retrieval of structural information when traditional NMR approaches fail. This particular project combines new isotopic labeling strategies developed in the Moremen group with two new types of NMR-observable structural data. One type comes from residual dipolar couplings of labeled 15N-1H pairs in various amide bonds. These give orientational constraints on bond vectors. The other type comes from long range paramagnetic spin relaxation perturbation of labeled sites by synthesized nitroxide-carrying analogs of protein ligands. These give long-range distance constraints between the bound ligand and the labeled sites. The sialyltransferase, ST6Gal-I, has been the primary target motivating this work. This transferase is essential to the synthesis of sialic acid terminated glycans on many glycoproteins, including those involved in the activation of B-cells. It was first cloned more than 18 years ago, but it continues to resist attempts at structural characterization by traditional means. A second target, the fucosyltransferase, FucT-III, is also structurally uncharacterized and equally important biologically. Both are representative of the many mammalian proteins that are best expressed in non-bacterial hosts.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 碳水化合物-蛋白质复合物的形成在涉及细胞与其环境相互作用的各种过程中非常重要。这些过程包括自然过程，例如细胞分化、细胞聚集和细胞信号传导；它们还包括病毒感染、恶性肿瘤和不必要的炎症等疾病过程。 缓和自然过程和抑制疾病过程的药物的开发始于寡糖-蛋白质相互作用的精确结构模型。由于寡糖-蛋白质复合物中缺乏短距离 NOE 接触，因此传统的核磁共振 (NMR) 方法很难获得这些模型。我们正在开发许多实验和分析工具，以便在传统核磁共振方法失败时检索结构信息。 这个特殊项目将 Moremen 小组开发的新同位素标记策略与两种新型 NMR 可观察结构数据相结合。 一种类型来自各种酰胺键中标记的 15N-1H 对的残余偶极耦合。 这些给出了键向量的方向约束。 另一种类型来自合成的携带硝基氧的蛋白质配体类似物对标记位点的长程顺磁自旋弛豫扰动。 这些给出了结合配体和标记位点之间的长距离距离限制。 唾液酸转移酶 ST6Gal-I 一直是推动这项工作的主要目标。这种转移酶对于许多糖蛋白（包括参与 B 细胞激活的糖蛋白）上唾液酸封端聚糖的合成至关重要。 它在 18 多年前首次被克隆，但它仍然抵制通过传统手段进行结构表征的尝试。第二个靶标岩藻糖基转移酶 FucT-III 在结构上也未表征，但在生物学上同样重要。 两者都是在非细菌宿主中表达最好的许多哺乳动物蛋白质的代表。