COMPUTER SIMULATION OF THE INFLUENZA M2 CHANNEL

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 跨细胞壁传输电荷的蛋白质的结构复杂性 在当代生物体中，这使得解剖分子结构变得非常困难。 他们的行动机制。因此，希望有一个蛋白质模型 它很小，有一个众所周知的结构基序，但与 更复杂的蛋白质的效率和控制。这导致了对 甲型流感病毒M2蛋白--一种小的同源四聚体电压门控离子 在脂质双层中自组装并以高浓度运输质子的通道 效率和选择性。每个单体由97个氨基酸组成， 单个跨膜结构域。此外，活跃的渠道 由仅含有25个氨基酸的亚组的合成肽重构 酸，包括跨膜区，没有特异性损失，或 效率该肽的氨基酸序列为Ser-Ser-Asp-Pro-Leu-。 Val-Val-Ala-Ala-Ser-Ile-Ile-Gly-Ile-Leu-His-Leu-Ile-Leu-Trp-Ile-Leu-Asp-Arg-Leu。 与grimicidin A相比，也许是最广泛研究的一种 质子通道，质子通过截断的M2通道的速率为 速度快了1000倍简单和高效的完美结合 使得M2不仅是理解简单肽如何 实现质子传输的高效率，但也具有吸引力和潜力 一个简单的质子泵的改造目标。 与短杆菌肽中质子转运的实验和理论研究一致，已经提出质子转运通过沿着跨越通道孔的水分子的瞬时链易位而发生。通道由四个组氨酸残基门控，这些组氨酸残基封闭管腔。这种门控机制可以解释为什么M2对碱金属离子是不可渗透的。然而，了解质子传导通过通道的完整过程需要更多的研究。半胱氨酸扫描诱变表明，孔衬残基的替换导致通道性质的大扰动，表明这些残基对通道效率是必不可少的。其他残基的身份起较小的作用。孔衬残基如何影响质子运输尚不清楚，因为这些残基都不是高极性的，也不能与水合氢离子形成特别强的氢键。 M2通道是pH门控的。在碱性和中性pH下，它似乎是封闭的。 低于5.5的pH（其也是组氨酸的pKa），通道打开， 观察到质子传输。因此，有人认为，四个中立的 组氨酸残基，而打开通道涉及 使一个（或多个组氨酸残基）质子化。Cross最近的NMR工作 同事们认为色氨酸残基接近于 组氨酸残基，也可能参与通道门控。他们 基于这些结果构建了一个模型结构（PDB名称1 NYJ）。 此外，他们最新的REDOR NMR结果表明，实际的门可能 由两个His-His+氢键对组成。根据这一结果， 认为在中性pH值下，两个组氨酸残基被质子化， 当三个（或四个）组氨酸在较低的温度下质子化时， 博士 最近，De格拉多实验室公布了M2在开放但封闭状态和封闭状态下的晶体结构，Schnell和Chou公布了封闭状态下的NMR结构。这些结构与1 NYJ模型有很大的不同，并且都没有表现出组氨酸残基之间的螺旋间氢键。除了形成通道门的His和Trp残基外，新结构都具有由束的N末端附近的瓦尔残基环形成的变窄。这被认为至少部分地形成了选择性过滤器;该区域中通道的变窄可以解释为什么通道可以传输质子而不能传输其他阳离子物种。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The structural complexity of proteins that transport charge across cell walls in contemporary organisms makes it extremely difficult to dissect the molecular mechanisms of their action. It is therefore desirable to have a protein model which is small and has a well known structural motif, yet operates with the efficiency and control of more complex proteins. This has led to the study of the Influenza A M2 protein -- a small, homotetrameric, voltage-gated ion channel which self-assembles in lipid bilayers and transports protons with high efficiency and selectivity. Each monomer is built of 97 amino acids and contains a single transmembrane domain. Additionally, active channels have been reconstituted from a synthetic peptide containing only a subset of 25 amino acids, including the transmembrane region, with no loss in specificity or efficiency. The sequence of amino acids in the peptide is Ser-Ser-Asp-Pro-Leu- Val-Val-Ala-Ala-Ser-Ile-Ile-Gly-Ile-Leu-His-Leu-Ile-Leu-Trp-Ile-Leu-Asp-Arg-Leu. Compared with grimicidin A, perhaps the most extensively studied model of a proton channel, the rate of proton transport across the truncated M2 channel is over 1000-fold faster. This remarkable combination of simplicity and efficiency makes M2 not only an excellent model for understanding how simple peptides can achieve high efficiency of proton transport but also an attractive, potential target for re-engineering a simple proton pump. In line with experimental and theoretical studies of proton transport in gramicidin, it has been suggested that proton transport occurs via translocation along a transient chain of water molecules that span the pore of the channel. The channel is gated by four histidine residues which occlude the lumen. This mechanism of gating can explain why M2 is impermeable to alkali ions. However, understanding the complete process of proton conductance through the channel requires additional studies. Cysteine scanning mutagenesis has shown that replacement of the pore-lining residues results in a large perturbation of the properties of the channel, indicating that these residues are essential for channel efficiency. The identities of other residues play a smaller role. How the pore-lining residues influence proton transport is not known, as none of these residues is highly polar or capable of forming particularly strong hydrogen bonds with the hydronium ion. The M2 channel is pH gated. At basic and neutral pH, it appears to be closed. Below a pH of 5.5 (which is also the pKa of histidine), the channel opens and proton transport is observed. It has therefore been argued that four neutral histidine residues from the gate, and that opening the channel involves protonating one (or more of the histidine residues). Recent NMR work by Cross and co-workers has suggested that the tryptophan residues are close to the histidine residues, and might also participate in channel gating. They constructed a model structure based on these results (PDB designation 1NYJ). Additionally, their newer REDOR NMR results indicate that the actual gate might consist of two His-His+ hydrogen bonding pairs. Based on this result, they have argued that at neultral pH, two of the histidine residues are protonated, and that the gate opens when three (or four) histidines become protonated at lower pH. Recently, crystal structures of M2 in an open, but blocked, state and in a closed state were published by the De Grado lab, and an NMR structure in a closed state was published by Schnell and Chou. These structures are quite different from the 1NYJ model, and neither exhibit the interhelical hydrogen bonding between Histidine residues. In addition to the His and Trp residues that appear to form the gate of the channel, the new structures both have a narrowing formed by a ring of Val residues near the N-terminal end of the bundle. This is thought to form, at least in part, the selectivity filter; the narrowing of the channel in this region could explain why the channel can transport protons but not other cationic species.