Solid-State NMR of Viral Fusion Peptides and Proteins

病毒融合肽和蛋白质的固态核磁共振

• 批准号: 8068086
• 负责人: David P Weliky
• 金额: $ 10.23万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8068086
• 关键词: Abbreviations; Acquired Immunodeficiency Syndrome; Affinity; Biochemical; Biological Assay; Biological Models; C-terminal; Catalysis; Cell Nucleus; Cell fusion; Cell membrane; Cells; Chemicals; Chimeric Proteins; Cholesterol; Circular Dichroism; Coupled; Coupling; Data; Dependence; Detergents; Development; Disease; Electron Spin Resonance Spectroscopy; Environment; Erythrocytes; Ethers; Glass; Glossary; Goals; HIV; Hemagglutinin; Infection; Influenza; Label; Link; Lipids; Liquid substance; Measurement; Membrane; Membrane Fusion; Membrane Proteins; Methods; Micelles; Molecular Conformation; Mutation; NMR Spectroscopy; Peptides; Play; Point Mutation; Proteins; Research; Resolution; Role; Sampling; Structural Models; Structure; Techniques; Tertiary Protein Structure; Testing; Therapeutic; Time; Torsion; Vesicle; Viral; Viral Fusion Proteins; Viral Proteins; Virus; analog; analytical ultracentrifugation; base; crosslink; dimer; influenzavirus; magnetic field; monomer; novel; protein aminoacid sequence; prototype; research study; solid state nuclear magnetic resonance

The long-term goal of the proposed research is to understand with atomic-resolution detail the basis for viral fusion protein-induced membrane fusion. Fusion of viral and target cell membranes is a key step in infection for many viruses important in disease and a detailed understanding of fusion mechanisms should aid development of anti-viral therapeutics whose mode of action is fusion inhibition. The proposed research focuses on the gp41 and the hemagglutinin HA2 fusion proteins of the human immunodeficiency virus (HIV) and the influenza virus (IFV), respectively, because of the significance of the diseases caused by these viruses and because these proteins serve as prototypes for other class I viral fusion proteins. There is particular emphasis on the "fusion peptide" (FP) domains of the fusion proteins, which represent ~20-residue apolar regions at the N-termini of the proteins. Numerous biochemical and biophysical studies have shown that the FP plays a key role in fusion and that chemically synthesized peptides with the.FP sequence can serve as useful model systems to understand some aspects of viral/target cell fusion. The long-term objectives will be achieved with four specific aims. Two of the aims focus on solid-state nuclear magnetic resonance (SSNMR) structural analysis of chemically cross-linked dimeric and trimeric HIV FPs in membranes and additional liquid-state NMR studies of these peptides in detergent micelles. The oligomeric topology of these C-terminal cross-linked peptides reflects the expected topology of FPs in the HIV gp41 fusion protein. The third aim focuses on: (a) determination of the insertion orientation of FPs in membranes using samples in which the membranes are oriented between stacked glass plates; and (b) probing FP insertion depths using SSNMR distance measurements between 13C nuclei in the FP and 31P and 19F nuclei in the lipid. The fourth specific aim focuses on SSNMR structural and insertion depth measurements for large HIV and IFV fusion protein domains which contain FPs. .

拟议研究的长期目标是通过原子分辨率详细了解 病毒融合蛋白诱导的膜融合。病毒和靶细胞膜的融合是关键步骤 许多对疾病很重要的病毒的感染以及对融合机制的详细了解应该 帮助开发其作用方式是融合抑制的抗病毒疗法。拟议的研究 专注于人类免疫缺陷病毒 (HIV) 的 gp41 和血凝素 HA2 融合蛋白 和流感病毒（IFV），因为它们引起的疾病的重要性 病毒，并且因为这些蛋白质可作为其他 I 类病毒融合蛋白的原型。有 特别强调融合蛋白的“融合肽”(FP) 结构域，它代表约 20 个残基 蛋白质 N 末端的非极性区域。大量的生化和生物物理研究表明 FP 在融合中起着关键作用，具有 FP 序列的化学合成肽可以 作为有用的模型系统来理解病毒/靶细胞融合的某些方面。 长期目标将通过四个具体目标来实现。其中两个目标集中在固态 化学交联二聚体和三聚体 HIV 的核磁共振 (SSNMR) 结构分析 膜中的 FP 以及洗涤剂胶束中这些肽的其他液态 NMR 研究。这 这些 C 端交联肽的寡聚拓扑反映了 FP 的预期拓扑 HIV gp41 融合蛋白。第三个目标侧重于：（a）确定 FP 的插入方向 使用其中膜在堆叠玻璃板之间定向的样品的膜； (b) 使用 FP 和 31P 中 13C 核之间的 SSNMR 距离测量来探测 FP 插入深度 和脂质中的 19F 核。第四个具体目标侧重于 SSNMR 结构和插入深度 包含 FP 的大型 HIV 和 IFV 融合蛋白结构域的测量​​。 。