Post-Expression Isotope Labeling: NMR Insights into Glycoprotein Structure

描述（由申请人提供）：核磁共振（NMR）光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在细菌宿主中容易表达的具有均匀同位素富集的可溶性蛋白。限制细菌表达使许多糖基化蛋白无法获得；它们代表了多达50%的人类编码蛋白质。提出的研究目的是为核磁共振提供同位素标记和分配方法，这并不局限于统一的同位素标记。这些分配策略将成为对一类特定的糖基化蛋白进行结构数据和功能分析的基础。我们计划研究的分配方法是通过化学和酶的方法在表达后同位素标记蛋白质。质谱（MS）将用于鉴定标记的肽，并协助分配核磁共振。我假设表达后，同位素标记策略，加上ms辅助核磁共振分配，将扩大核磁共振光谱的范围，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，这是以前无法实现的。该独立途径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甲基化模型蛋白对蛋白质和配体结合位点进行结构表征。作为一名独立研究者，她的具体目标是应用酶同位素标记方法进行谷氨酰胺和聚糖标记，开发核磁共振实验进行分配，并将同位素标记和分配策略应用于Notch1和其他生物学相关靶点的结构域。

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.