NEW NMR METHODOLOGY FOR CHARACTERIZING CARBOHYDRATE-PROTEIN INTERACTIONS

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 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是重要的。这些过程包括自然过程，如细胞分化、细胞聚集和细胞信号传导;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。开发药物以缓和自然过程和抑制疾病过程始于寡糖-蛋白质相互作用的精确结构模型。由于寡糖-蛋白质复合物中缺少短距离NOE接触，这些模型很难通过传统的核磁共振（NMR）方法得到。我们正在开发一些实验和分析工具，当传统的NMR方法失败时，可以检索结构信息。 这个特殊的项目结合了Moremen小组开发的单一氨基酸标记策略和两种新类型的NMR可观察的结构数据。 一种类型来自各种酰胺键中标记的15 N-1H对的残余偶极偶联。 这些给键矢量的取向约束。 另一种类型来自合成的蛋白质配体的携带氮氧自由基的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程距离约束。 唾液酸转移酶，ST 6 Gal I，一直是推动这项工作的主要目标。这种转移酶在超过17年前首次克隆（Weinstein，Ujita，Paulson，Fed.继续，44，690-690，1985），但它继续抵制通过传统手段进行结构表征的尝试。它在B细胞的活化等方面具有重要意义，但它也代表了许多在非细菌宿主中表达最好的重要哺乳动物蛋白质。

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 a single amino acid labeling strategy 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 was first cloned more than 17 years ago (Weinstein, Ujita, Paulson, Fed. Proceed., 44, 690-690, 1985), but it continues to resist attempts at structural characterization by traditional means. It is of importance in the activation of B-cells among other things, but it also is representative of many important mammalian proteins that are best expressed in non-bacterial hosts.