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.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 碳水化合物-蛋白质复合体的形成在涉及细胞与其环境相互作用的各种过程中是重要的。这些过程包括自然的过程，如细胞分化、细胞聚集和细胞信号传递；它们还包括疾病过程，如病毒感染、恶性肿瘤和不必要的炎症。用来缓和自然过程和抑制疾病过程的药物的开发始于寡糖-蛋白质相互作用的准确结构模型。由于寡糖-蛋白质复合体中缺少短程NOE接触，这些模型很难用传统的核磁共振方法得到。我们正在开发一些实验和分析工具，当传统的核磁共振方法失败时，这些工具可以检索结构信息。这个特殊的项目结合了莫尔门小组开发的新的同位素标记策略和两种新类型的核磁共振可观测结构数据。 一种类型来自标记的15N-1H对在不同的酰胺键中的残余偶极耦合。这给出了键矢的取向约束。另一类来自合成的含氮类似物对标记位点的长程顺磁自旋弛豫扰动。这些都给出了结合配体和标记位点之间的长距离限制。唾液酸基转移酶ST6Gal-I一直是推动这项工作的主要靶点。这种转移酶对合成许多糖蛋白上的唾液酸端接糖链是必不可少的，包括那些参与B细胞激活的糖蛋白。它最初是在18年前克隆的，但它继续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，在结构上也是未知的，在生物学上也同样重要。这两种蛋白都代表了在非细菌宿主中最好表达的许多哺乳动物蛋白。