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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

基本信息

批准号：
7667545
负责人：
Megan Alane Macnaughtan
金额：
$ 24.85万
依托单位：
LOUISIANA STATE UNIV A&M COL BATON ROUGE
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-09-15 至 2011-08-17
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7667545
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 protein structure research study tool

项目摘要

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.

核磁共振（NMR）波谱是表征物质结构的有力工具