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

描述（由申请方提供）：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主要的公共卫生问题，无论是在其每年的死亡人数和它的潜力，造成毁灭性的流行病。M2蛋白是病毒的基本成分，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，其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在以前的流感大流行中有效预防。M2蛋白的N-末端胞外结构域在所有人类甲型流感病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。要全面了解M2的功能和流感的生物学特性，就必须了解这种蛋白质的结构。此外，作为一个相对较小的膜蛋白，M2蛋白是一个实验上易于处理的和有价值的生物物理模型，了解离子通道功能，并作为一个试验场，探索膜蛋白折叠的基本原理。 我们的策略研究构象的M2涉及合成纳入自旋标记在一系列网站内的蛋白质，重组的标记蛋白质到脂质膜和测量的电子顺磁共振（EPR）的光谱特性的标签。从EPR研究中可以提取几种类型的结构信息：跨越蛋白质序列的标记迁移率模式可用于表征蛋白质折叠的地形区域，标记与不同双层/水溶性的顺磁性试剂碰撞的可接近性也可用于确定膜中自旋标记残基的深度，并且可以通过测量自旋-自旋耦合来确定两个标签之间的距离。 将依次检查M2蛋白的跨膜结构域、C-末端胞质结构域和N-末端胞外结构域。EPR数据将在各种脂质双层组合物中以及在抗病毒药物金刚烷胺存在下收集。M2通道是pH门控的，将在几个pH值下检查自旋标记蛋白，以探测与通道门控相关的构象变化。

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.