Determining the Conformation of the Influenza A M2 Protein using EPR Spectroscopy

使用 EPR 光谱测定甲型流感 M2 蛋白的构象

• 批准号: 7364732
• 负责人: KATHLEEN P HOWARD
• 金额: $ 20.07万
• 依托单位: SWARTHMORE COLLEGE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7364732
• 关键词: Amantadine; Amino Acid Sequence; Antiviral Agents; Binding; Biology; C-terminal; Cessation of life; Classification; Coupling; Cytoplasmic Tail; DNA Sequence Rearrangement; Data; Dependence; Depth; Electron Spin Resonance Spectroscopy; Elements; Employee Strikes; Environment; Extracellular Domain; Goals; Helix (Snails); Homo; Human; Influenza; Influenza A virus; Influenza A virus M2 protein; Ion Channel; Label; Lateral; Length; Lipid Bilayers; Lipids; M2 protein; Measurement; Measures; Membrane; Membrane Lipids; Membrane Proteins; Methods; Modeling; Molecular Conformation; N-terminal; Pattern; Peptide Sequence Determination; Peptides; Pharmaceutical Preparations; Phase; Pore Proteins; Property; Prophylactic treatment; Proteins; Protons; Public Health; Range; Reagent; Research; Rimantadine; Series; Site; Solubility; Spin Labels; Structural Models; Structure; System; Testing; Thick; Transmembrane Domain; Virion; Virus; Virus Replication; Work; aqueous; ear helix; influenza virus vaccine; insight; interest; pandemic disease; pandemic influenza; pressure; protein folding; protein structure; reconstitution; tool

DESCRIPTION (provided by applicant): The overall goal of this research is to determine the conformation of the M2 protein from the influenza A virus. Influenza A virus is a major public health concern, both in its annual death toll and its potential to cause devastating pandemics. The M2 protein is an essential element of the virus and serves as a proton channel. The channel is the target of the antiviral drugs amantadine and rimantadine, which block proton channel activity and inhibit virus replication. These antiviral drugs have proven effective for prophylaxis in prior influenza pandemics. The N-terminal extracellular domain of the M2 protein has striking sequence conservation across all human influenza A strains and is being developed as a target for a universal flu vaccine. A full understanding of the function of M2 and the biology of influenza relies on understanding the structure of this protein. Furthermore, as a relatively small membrane protein, the M2 protein is an experimentally tractable and valuable biophysical model for understanding ion channel function and as a testing ground for exploring the basic principles of membrane protein folding. Our strategy for studying the conformation of M2 involves synthetically incorporating spin labels at a series of sites within the protein, reconstituting the labeled protein into lipid membranes and measuring the electron paramagnetic resonance (EPR) spectral properties of the labels. Several types of structural information can be extracted from EPR studies: patterns of label mobilities across a protein sequence can be used to characterize topographic regions of a protein fold, accessibility of a label to collision with paramagnetic reagents of varying bilayer/aqueous solubility can also be used to determine the depth of a spin-labeled residue in the membrane, and the distance between two labels can be determined from the measurement of spin-spin couplings. The transmembrane domain, C-terminal cytoplasmic domain and the N-terminal extracellular domain of the M2 protein will each be examined in turn. EPR data will be collected in a variety of lipid bilayer compositions and in the presence of the antiviral drug amantadine. The M2 channel is pH-gated and the spin-labeled protein will be examined at several pH values to probe for conformational changes related to channel gating.

描述（由申请人提供）：本研究的总体目标是确定甲型流感病毒M2蛋白的构象。甲型流感病毒是一个重大的公共卫生问题，无论是其每年的死亡人数还是其造成毁灭性大流行的潜力。M2蛋白是病毒的基本元素，并作为质子通道。该通道是抗病毒药物金刚烷胺和金刚乙胺的靶点，它们阻断质子通道活性并抑制病毒复制。这些抗病毒药物已被证明对以前流感大流行的预防有效。M2蛋白的n端胞外结构域在所有人类甲型流感毒株中具有惊人的序列保守性，并且正在开发作为通用流感疫苗的靶点。充分了解M2的功能和流感生物学依赖于了解这种蛋白质的结构。此外，M2蛋白作为一种相对较小的膜蛋白，是一种实验上易于处理和有价值的生物物理模型，可以用于理解离子通道功能，并作为探索膜蛋白折叠基本原理的试验场。