Post-Expression Isotope Labeling: NMR Insights into Glycoprotein Structure

核磁共振波谱是表征化合物结构的有力工具 并深入了解它们的功能。然而，通过核磁共振解析的结构已经严重地 偏向于易于在具有统一同位素的细菌宿主中表达的可溶性蛋白质 浓缩物。对细菌表达的限制使许多糖基化蛋白无法获得；这些 代表高达50%的编码人类蛋白质。拟议研究的目标是提供 核磁共振的同位素标记和分配方法，不限于统一的同位素标记。 这些分配策略将成为对 糖基化蛋白质的特定类别。我们计划研究的分配方法是用同位素 通过化学和酶方法标记蛋白质的表达后。将使用质谱仪(MS) 鉴定标记的多肽并协助指定核磁共振波谱。我假设这个后置表达， 同位素标记策略，再加上MS辅助核磁共振指定，将扩大 核磁共振光谱学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，以前是无法获得的。在此期间的具体目标是 本指导阶段的独立之路奖是为提高MS的分辨率而协助的 还原~(13)C甲基化赖氨酸的核磁共振指认策略及蛋白质结构表征 以及使用还原13C甲基化模型蛋白的配体结合位点。具体目标应追求为 一名独立的研究人员将应用酶同位素标记方法来检测谷氨酰胺和多糖 标记，开发用于分配的核磁共振实验，并应用同位素标记和分配策略 来自Notchl和其他生物相关靶标的结构域。 了解蛋白质的结构和功能对于疾病的诊断、治疗和 预防。能够将蛋白质的特性限制在可溶的、非糖基化蛋白质的子集上 我们将从研究中排除一类占人类所有蛋白质高达50%的蛋白质， 以及许多与疾病相关的蛋白质。拟议的研究将引入新的工具来 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.