Sparse NMR Labeling Approach to Glycoprotein Structure and Function

糖蛋白结构和功能的稀疏 NMR 标记方法

• 批准号: 9810830
• 负责人: JAMES H. PRESTEGARD
• 金额: $ 30.2万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9810830
• 关键词: Address; Adopted; Affect; Amides; Amino Acids; Binding Proteins; Biological; Biology; Bos taurus structural-GP protein; Cell Culture Techniques; Chemicals; Collection; Communities; Computer Simulation; Computer software; Crystallization; Data; Data Analyses; Data Set; Development; Dimensions; Disease; Equilibrium; Exclusion; Genetic Programming; Glycoproteins; Goals; HMQC; Hand; Health; Homology Modeling; Human; Isotope Labeling; Isotopes; Label; Magnetism; Mammalian Cell; Manuals; Measurement; Measures; Methods; Modeling; Motion; Mutation; Nuclear; Nuclear Magnetic Resonance; Output; Physiology; Procedures; Process; Production; Program Efficiency; Proteins; Protocols documentation; Protons; Relaxation; Research Personnel; Residual state; Resolution; Route; Scheme; Site; Source; Structural Models; Structural Protein; Structure; Surface; System; Technology; Time; Validation; Weight; Work; X-Ray Crystallography; base; computer generated; design; experimental study; glycoprotein structure; glycosylation; improved; methyl group; mutant; off-label use; pathogen; preservation; prevent; programs; protein protein interaction; protein structure; simulation

SUMMARY Glycoproteins represent a class of mammalian proteins that presents challenges for structural and functional characterization, particularly if the native glycosylation, which affects structure, stability and interaction with other molecules, is to be preserved. The best route to proteins with native glycosylation is expression in mammalian cell cultures. Unfortunately, for X-ray crystallography, this produces proteins with heterogenous glycosylation, often preventing formation of suitable crystals. For traditional nuclear magnetic resonance (NMR) methods, this forces use of expensive substrates for isotopic labeling and prohibits the perdeuteration often required to maintain resolution for larger proteins. The investigators involved in this proposal have worked together to develop an efficient mammalian cell expression system that produces proteins sparsely labeled using a restricted set of less expensive isotope enriched amino acids and maintains resolution without the aid of perdeuteration. This has been accompanied by the development of resonance assignment programs and data analysis protocols that allow structural and functional characterization from basic, high sensitivity, two-(and three-)dimensional NMR experiments. This project is designed to turn those developments into an integrated protocol that can be adopted by an expanded community of users. It centers on the refinement of a software package that accomplishes NMR resonance assignment of sparsely labeled proteins. It will be bolstered by extensive validation of program output, introduction of new data types and data analysis methods, and extension of program capabilities to the refinement of computer-generated models for protein structure. The potential impact will be a new route to structure and function studies of a class of proteins intimately involved with human physiology and disease.

概括 糖蛋白代表一类哺乳动物蛋白质，对结构和蛋白质结构提出了挑战。 功能表征，特别是天然糖基化，它会影响结构、稳定性和 与其他分子的相互作用，将被保留。获得具有天然糖基化的蛋白质的最佳途径是 在哺乳动物细胞培养物中表达。不幸的是，对于 X 射线晶体学来说，这会产生具有 异质糖基化，通常会阻止合适晶体的形成。对于传统核磁 共振（NMR）方法，这迫使使用昂贵的底物进行同位素标记并禁止 通常需要全氘化来维持较大蛋白质的分辨率。涉案调查人员 该提案共同开发了一种高效的哺乳动物细胞表达系统，可产生 使用一组有限的较便宜的同位素富集氨基酸稀疏地标记蛋白质，并保持 无需置换的帮助即可解决。这伴随着共振的发展 分配程序和数据分析协议，允许结构和功能表征 基本、高灵敏度、二维（和三维）核磁共振实验。该项目旨在将这些 开发成一个可以被扩大的用户社区采用的集成协议。它集中 关于完成稀疏标记的 NMR 共振分配的软件包的改进 蛋白质。它将通过对程序输出的广泛验证、新数据类型和数据的引入来支持 分析方法，以及程序功能的扩展，以细化计算机生成的模型 蛋白质结构。潜在的影响将是一类结构和功能研究的新途径 与人类生理和疾病密切相关的蛋白质。