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有力工具。然而，通过NMR解析的结构严重偏向于可溶性蛋白质，这些蛋白质可以在具有均匀同位素富集的细菌宿主中容易地表达。对细菌表达的限制使得许多糖基化蛋白无法获得;这些糖基化蛋白占编码的人类蛋白的50%。提出的研究的目的是提供同位素标记和分配方法的NMR，这是不限于统一的同位素标记。这些分配策略将成为一个特定类别的糖基化蛋白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促方法对表达后的蛋白质进行同位素标记。质谱法（MS）将用于鉴别标记的肽，并协助分配NMR共振。我假设，表达后，同位素标记策略，再加上MS辅助NMR分配，将扩大NMR光谱的范围，包括新的蛋白质类，并允许表征蛋白质-蛋白质和碳水化合物介导的相互作用，以前无法访问。该独立之路奖指导阶段的具体目标是提高还原13 C-甲基化赖氨酸的MS辅助NMR分配策略的分辨率，并使用还原13 C-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。作为一个独立的研究者追求的具体目标是应用酶的同位素标记方法的谷氨酰胺和聚糖标记，开发NMR实验分配，并应用同位素标记和分配策略，从Notch 1和其他生物相关的目标域。 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，我们将从研究中排除一类占所有人类蛋白质50%的蛋白质和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，特别强调糖蛋白。

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.