Membrane Protein Structure Using Evolutionary Couplings and Sparse NMR Data

使用进化耦合和稀疏 NMR 数据的膜蛋白结构

• 批准号: 9978825
• 负责人: GAETANO T MONTELIONE
• 金额: $ 51.98万
• 依托单位: RENSSELAER POLYTECHNIC INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9978825
• 关键词: 3-Dimensional; Address; Advisory Committees; Algorithmic Software; Algorithms; Antibiotics; Base Sequence; Benchmarking; Bioinformatics; Biological; Complex; Computing Methodologies; Coupling; Cryoelectron Microscopy; Data; Detergents; Electron Microscopy; Ensure; Environment; Enzymes; Equilibrium; Escherichia coli; Future; Genome; Goals; Homeostasis; Hybrids; Integral Membrane Protein; Isotope Labeling; Isotopes; Ligand Binding; Lipoproteins; Literature; Membrane; Membrane Proteins; Methods; Micelles; Modeling; Molecular Conformation; Multi-Drug Resistance; Mutation; NOESY; National Institute of Allergy and Infectious Disease; Nuclear Magnetic Resonance; Phylogenetic Analysis; Preparation; Production; Program Development; Proteins; Protocols documentation; Reproducibility; Research; Resolution; Resources; Sampling; Sequence Alignment; Structural Models; Structure; System; Testing; Time; United States National Institutes of Health; Validation; X-Ray Crystallography; biodefense; cryogenics; data quality; design; dimer; drug development; drug discovery; improved; innovation; maltose-binding protein; method development; molecular modeling; nanodisk; pathogen; pathogenic bacteria; periplasm; priority pathogen; programs; protein structure; receptor; reconstitution; screening; solid state nuclear magnetic resonance; structural biology; success; three dimensional structure; three-dimensional modeling; tool

PROJECT SUMMARY Integral Membrane Proteins (IMPs) include many biomedically-important gate keepers, receptors, transporters, homeostasis regulators, and potential drug discovery targets. Three-dimensional (3D) structure determination of IMPs by X-ray crystallography, cryo-electron microscopy (cryo-EM), or Nuclear Magnetic Resonance (NMR) methods remains a major challenge for structural biology. While NMR can generally provide accurate 3D structures of small soluble proteins, structure determination by solution NMR of IMPs, prepared in stabilizing membrane-mimicking environments which generally require 2H,13C,15N-enrichment of the IMP, can be quite challenging. Evolutionary couplings (ECs), evolutionarily-correlated mutations derived from multiple sequence alignments, can also be used to provide information about native residue pair contacts, and to model the 3D structures of IMPs. Combining EC and NMR data provides a powerful approach for overcoming incompleteness of NMR NOESY data obtained for perdeuterated IMP samples, and the challenges in identifying true native protein structure contacts from the phylogenetic EC analysis. In particular, inter-helical contact information that is difficult to obtain for perdeuterated IMPs by NMR is well represented in the sequence co-variance EC data. Our goals are to develop a robust, reproducible, and fully automated EC-NMR platform suitable for accurate and reliable structure determination of IMPs, particularly α-helical IMPs, and apply these methods for 3D structure analysis of biomedically- important IMPs. EC-NMR will be further developed using β-barrel and α-helical IMPs of known structure, and then applied to studies of IMPs of unknown structure selected from designated NIH NIAID priority pathogenic bacteria. We will (i) further develop and apply the Single Protein Production (SPP) method for producing isotope-enriched IMPs in E. coli, (ii) implement a micro-scale NMR screening pipeline for IMP sample optimization, (iiii) rigorously and comprehensively address the question of how EC and NOESY data quality and quantity correlate with the accuracy of EC-NMR structures, (iv) design improved algorithms for structure determination of IMPs combining ECs and NMR data, and (v) develop tools for validation of IMP structures determined by both conventional NMR and EC-NMR methods. Advanced molecular modeling methods will be implemented to improve accuracy of EC-NMR structures. ECs will also be combined with NMR data to identify and determine structures of multiple “native states” of proteins. This study will expand the range of proteins that can be studied by NMR, provide more accurate structural and dynamic information than can be obtained with existing methods, and provide fundamental structural information needed for future antibiotic drug development targeted to high-priority pathogens.

项目摘要 整合膜蛋白（IMP）包括许多生物医学上重要的门控蛋白，受体， 转运蛋白、稳态调节剂和潜在的药物发现靶点。三维（3D） 通过X射线晶体学、冷冻电子显微镜（cryo-EM）或 核磁共振（NMR）方法仍然是结构生物学的主要挑战。 虽然NMR通常可以提供小的可溶性蛋白质的精确3D结构，但结构 通过溶液NMR测定在稳定膜模拟环境中制备的IMP 这通常需要IMP的2 H、13 C、15 N富集，可能相当具有挑战性。进化 偶联（EC），来自多个序列比对的进化相关突变，可以 也可用于提供有关天然残基对接触的信息，并对3D IMP的结构。结合EC和NMR数据提供了一种强大的方法， 全氘代IMP样品NMR NOESY数据的不完整性，以及 从所述系统发育EC分析鉴定真正的天然蛋白质结构接触。特别是， 螺旋间的接触信息，这是很难获得全氘代IMP的NMR是很好的 表示在序列协方差EC数据中。我们的目标是开发一个强大的，可重复的， 和全自动EC-NMR平台，适用于准确可靠的结构测定， IMP，特别是α-螺旋IMP，并将这些方法应用于生物医学- 重要的IMP。EC-NMR将进一步发展使用已知的β-桶和α-螺旋IMP 结构，然后应用于从指定NIH中选择的未知结构的IMP的研究 NIAID优先致病菌。我们将（i）进一步开发和应用单一蛋白质生产 (SPP)在大肠杆菌中生产同位素富集的IMP的方法。大肠杆菌，（ii）实现微尺度NMR IMP样品优化的筛选管道，（iii）严格和全面地解决 EC和NOESY数据的质量和数量如何与EC-NMR的准确性相关的问题 结构，（iv）设计改进的算法，用于结合EC和 NMR数据，以及（v）开发用于验证由两种常规方法确定的IMP结构的工具 NMR和EC-NMR方法。先进的分子建模方法将被实施，以改善 EC-NMR结构的准确性。EC还将与NMR数据相结合， 蛋白质的多种“天然状态”的结构。这项研究将扩大蛋白质的范围， 通过核磁共振研究，提供比可以获得的更准确的结构和动力学信息 与现有的方法，并提供未来抗生素所需的基本结构信息， 针对高优先级病原体的药物开发。