Post-Expression Isotope Labeling: NMR Insights into Glycoprotein Structure

核磁共振（NMR）光谱是表征化合物结构的有力工具。 蛋白质并深入了解它们的功能。然而，通过NMR解析的结构已经被严重地 偏向于可溶性蛋白质，其可以容易地在具有均匀同位素的细菌宿主中表达 丰富。对细菌表达的限制使许多糖基化蛋白无法获得;这些糖基化蛋白质在细菌中表达。 代表了多达50%的编码人类蛋白质。本研究的目的是提供 用于NMR的同位素标记和分配方法，其不限于均匀的同位素标记。 这些分配策略将成为结构数据和功能分析的基础， 特定类别的糖基化蛋白质。我们计划研究的分配方法是 通过化学和酶促方法标记表达后蛋白质。将使用质谱法（MS） 以鉴定标记的肽并协助NMR共振的分配。I hypothesize假设that postexpression后， 同位素标记策略，加上MS辅助NMR分配，将扩大范围， NMR光谱包括新的蛋白质类别，并允许表征蛋白质-蛋白质和 碳水化合物介导的相互作用是以前无法达到的。会议期间的具体目标 这条独立之路奖的指导阶段是为了提高MS辅助的解决方案， 还原性13 C-甲基化赖氨酸的NMR分配策略和结构表征蛋白质 和配体结合位点。具体目标如下： 一个独立的研究者将应用酶同位素标记法对谷氨酰胺和聚糖 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 来自Notchl和其他生物学相关靶标的结构域。 了解蛋白质的结构和功能对于疾病的诊断、治疗和预防是非常重要的。 预防具有表征蛋白质的能力，仅限于可溶性非糖基化蛋白质的子集 我们会从研究中排除一类占人类蛋白质50%的蛋白质， 和许多与疾病相关的蛋白质。拟议的研究将引入新的工具， 描述蛋白质的结构，特别强调糖蛋白。

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 postexpression, 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 Notchl 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.