Post-Expression Isotope Labeling: NMR Insights into Glycoprotein Structure

表达后同位素标记：NMR 洞察糖蛋白结构

• 批准号: 7318650
• 负责人: Megan Alane Macnaughtan
• 金额: $ 8.96万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2008-08-17
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7318650
• 关键词: Accounting; Acids; Amino Acids; Award; Bacteria; Binding Sites; Biological Assay; Carbohydrates; Chemicals; Class; Code; Collection; Complex; Coupled; Coupling; Cyclotrons; Data; Development; Diagnosis; Digestion; Disease; Disease Progression; Electrospray Ionization; Enzymes; Epidermal Growth Factor; Fourier Transform; Glutamine; Glycoproteins; Goals; HMQC; Human; Indium; Ions; Isotope Labeling; Isotopes; Label; Left; Ligand Binding; Link; Liquid Chromatography; Lysine; Mass Spectrum Analysis; Measurement; Mediating; Membrane Proteins; Mentors; Methodology; Methods; Modeling; NMR Spectroscopy; Nuclear Magnetic Resonance; Oligosaccharides; Pathway interactions; Pepsin A; Peptides; Phase; Polysaccharides; Prevention; Production; Proteins; Relaxation; Research; Research Personnel; Residual state; Resolution; ST6Gal I; Selenomethionine; Serum; Sialic Acids; Sialyltransferases; Signal Transduction; Site; Spectrometry; Structure; System; Techniques; Testing; Transferrin; Transglutaminases; X-Ray Crystallography; amino group; base; glycoprotein structure; glycosylation; improved; insight; methyl group; notch protein; novel strategies; nuclear Overhauser enhancement; programs; protein structure; research study; tool

DESCRIPTION (provided by applicant): Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for characterizing the structure of proteins and gaining insight into their function. However, structures solved by NMR have been heavily biased toward soluble proteins that can be easily expressed in bacterial hosts with uniform isotope enrichment. Restriction to bacterial expression leaves many glycosylated proteins inaccessible; these represent as much as 50% of coded human proteins. The objective of the proposed research is to provide isotope-labeling and assignment methodologies for NMR, which are not limited to uniform isotope labeling. These assignment strategies will become the underpinnings for structural data and functional assays on a specific class of glycosylated proteins. The assignment approach we plan to investigate is to isotopically label proteins post-expression via chemical and enzymatic methods. Mass spectrometry (MS) will be used to identify the peptides labeled and assist in the assignment of NMR resonances. I hypothesize that post-expression, isotope labeling strategies, coupled with MS-assisted NMR assignment, will expand the scope of NMR spectroscopy to include new classes of proteins and allow characterization of protein-protein and carbohydrate-mediated interactions that have been previously inaccessible. The specific aims during the mentored phase of this Pathway to Independence Award are to improve the resolution of the MS-assisted NMR assignment strategy for reductively 13C-methylated lysines and to structurally characterize proteins and ligand binding sites using reductively 13C-methylated model proteins. Specific aims to be pursued as an independent investigator are to apply enzymatic isotope labeling methods for glutamine and glycan labeling, develop NMR experiments for assignment, and apply isotopic labeling and assignment strategies to domains from Notch1 and other biologically relevant targets. Understanding the structure and function of a protein is important to disease diagnosis, treatment, and prevention. With the ability to characterize proteins limited to a subset of soluble, non-glycosylated proteins, we would exclude from study a class of proteins that accounts for as much as 50% of all human proteins and many proteins specifically linked to disease. The proposed research will introduce new tools to characterize the structure of proteins with particular emphasis on glycoproteins.

描述（由申请人提供）：核磁共振（NMR）波谱是表征蛋白质结构和深入了解其功能的强大工具。然而，核磁共振解析的结构严重偏向于可溶性蛋白质，这些蛋白质可以在细菌宿主中轻松表达，并具有均匀的同位素富集。对细菌表达的限制使得许多糖基化蛋白质无法接近；这些代表了多达 50% 的人类编码蛋白质。本研究的目的是为 NMR 提供同位素标记和分配方法，不仅限于统一同位素标记。这些分配策略将成为特定类别糖基化蛋白质的结构数据和功能测定的基础。我们计划研究的分配方法是通过化学和酶法对表达后的蛋白质进行同位素标记。质谱 (MS) 将用于鉴定标记的肽并协助 NMR 共振的分配。我假设表达后、同位素标记策略与 MS 辅助 NMR 分配相结合，将扩大 NMR 波谱的范围，以包括新类别的蛋白质，并允许对以前无法实现的蛋白质-蛋白质和碳水化合物介导的相互作用进行表征。该独立之路奖指导阶段的具体目标是提高还原 13C 甲基化赖氨酸的 MS 辅助 NMR 分配策略的分辨率，并使用还原 13C 甲基化模型蛋白质对蛋白质和配体结合位点进行结构表征。作为独立研究者所追求的具体目标是应用酶促同位素标记方法进行谷氨酰胺和聚糖标记，开发用于分配的 NMR 实验，并将同位素标记和分配策略应用于 Notch1 和其他生物学相关目标的域。 了解蛋白质的结构和功能对于疾病的诊断、治疗和预防非常重要。由于能够表征仅限于可溶性非糖基化蛋白质子集的蛋白质，我们将从研究中排除一类占所有人类蛋白质 50% 的蛋白质以及许多与疾病特异性相关的蛋白质。拟议的研究将引入新的工具来表征蛋白质的结构，特别是糖蛋白。