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: 7157007
• 负责人: David P Weliky
• 金额: $ 27.73万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-01 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7157007
• 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; Class; Coupled; Coupling; Data; Dependence; Depth; Detergents; Development; Disease; Electron Spin Resonance Spectroscopy; Environment; Erythrocytes; Ethers; Ethyl Ether; Glass; Glossary; Goals; HIV; Hemagglutinin; Human Parainfluenza Virus 1; Infection; Influenza; Intermediate Filament Proteins; Label; Link; Lipids; Liquid substance; Measurement; Membrane; Membrane Fusion; Membrane Proteins; Methods; Micelles; Molecular Conformation; Mutation; NMR Spectroscopy; Nuclear Magnetic Resonance; Peptides; Play; Point Mutation; Proteins; Rate; 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

DESCRIPTION (provided by applicant): 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 approximately 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.

描述（由申请人提供）：拟议研究的长期目标是以原子分辨率详细了解病毒融合蛋白诱导膜融合的基础。病毒和靶细胞膜的融合是许多在疾病中重要的病毒感染的关键步骤，对融合机制的详细了解应该有助于开发作用方式为融合抑制的抗病毒治疗方法。拟议的研究分别集中在人类免疫缺陷病毒（HIV）和流感病毒（IFV）的gp 41和血凝素HA 2融合蛋白上，因为这些病毒引起的疾病的重要性，并且因为这些蛋白质作为其他I类病毒融合蛋白的原型。特别强调融合蛋白的“融合肽”（FP）结构域，其代表蛋白质N-末端的约20个残基的非极性区域。许多生物化学和生物物理学研究表明，FP在融合中起着关键作用，化学合成的FP序列肽可以作为有用的模型系统，以了解病毒/靶细胞融合的某些方面。将通过四个具体目标实现长期目标。两个目标集中在固态核磁共振（SSNMR）的化学交联的二聚体和三聚体的HIV FP在膜和额外的液态NMR研究这些肽在洗涤剂胶束的结构分析。这些C-末端交联肽的寡聚拓扑结构反映了HIV gp 41融合蛋白中FP的预期拓扑结构。第三个目标侧重于：（a）使用样品测定FP在膜中的插入取向，其中膜在堆叠的玻璃板之间取向;和（B）使用FP中的13 C核与脂质中的31 P和19 F核之间的SSNMR距离测量来探测FP插入深度。第四个具体目标集中在SSNMR结构和插入深度测量的大型HIV和IFV融合蛋白结构域，其中包含FP。