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.

总结 糖蛋白代表一类哺乳动物蛋白，其对结构和功能提出了挑战。 功能表征，特别是如果天然糖基化，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，这产生了具有 异源糖基化，经常阻止合适晶体的形成。对于传统的核磁共振 在使用核磁共振（NMR）方法时，这迫使使用昂贵的底物进行同位素标记，并禁止了同位素标记。 通常需要全氘代以维持较大蛋白质的分辨率。参与此事的调查人员 已经有人提出共同开发有效的哺乳动物细胞表达系统， 使用有限的一组较便宜的同位素富集氨基酸稀疏标记的蛋白质， 不借助过氘化的解决方案。这一直伴随着共振的发展 分配程序和数据分析协议，允许结构和功能表征， 基本的、高灵敏度的、二维（和三维）NMR实验。这个项目的目的是把那些 发展成为一个可被扩大的用户群体采用的综合议定书。It中心 改进的软件包，完成NMR共振分配稀疏标记的 proteins.它将通过广泛验证程序输出、引入新的数据类型和数据 分析方法，并将程序功能扩展到计算机生成的模型的细化， 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。