COMPUTER SIMULATION OF THE INFLUENZA M2 CHANNEL

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 跨细胞壁运输电荷的蛋白质的结构复杂性 使得在当代生物体中解剖分子极其困难 它们的作用机制。因此，希望有一种蛋白质模型 它很小，有一个众所周知的结构主题，但它与 更复杂蛋白质的效率和控制。这导致了对 甲型流感M2蛋白--一种小的、同分异构体、电压门控离子 在脂质双层中自组装并运输高密度质子的通道 效率和选择性。每个单体由97个氨基酸组成，含有 一个单一的跨膜结构域。此外，活动的通道已经 由仅含有25个氨基的合成肽重组而成 酸，包括跨膜区，而不损失特异性或 效率。其氨基酸序列为：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. 与格里米菌素A相比，可能是研究最广泛的一种 质子通道，通过截断的M2通道的质子传输速率为 速度提高1000倍以上。这种简单和高效的非凡结合 使M2不仅是了解简单多肽如何 实现高效率的质子传输，也有诱人的潜力 目标是重新设计一个简单的质子泵。 与实验和理论研究相一致的是，氢化甘草素中的质子传输是通过沿跨越通道孔道的水分子的瞬时链进行传输进行的。该通道由四个组氨酸残基控制，它们阻塞管腔。这种门控机制可以解释M2对碱性离子不渗透的原因。然而，要了解通过该通道的质子电导的完整过程，需要进行额外的研究。半胱氨酸扫描突变表明，孔衬残基的替换会导致通道性质的很大扰动，表明这些残基对通道效率是必不可少的。其他残基的身份起到的作用较小。孔衬残基如何影响质子传输尚不清楚，因为这些残基都不是高极性的，也不能与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。 在pH值5.5(也是组氨酸的pKa)以下，该通道打开并 观察到了质子输运。因此，有人认为，四个中立 门上的组氨酸残留物，打开通道涉及 质子化一个(或多个组氨酸残基)。克罗斯最近的核磁共振工作 同事们表示，色氨酸残留量接近 组氨酸残基，也可能参与通道门控。他们 根据这些结果构建了一个模型结构(PDB编号1NYJ)。 此外，他们新的Redor核磁共振结果表明，实际的星门可能 由两个His-His氢键对组成。根据这一结果，他们有 认为在中性pH下，两个组氨酸残基被质子化，并且 当三个(或四个)组氨酸在较低的位置质子化时，门就会打开 PH值。 最近，De Grado实验室公布了M2在开放但封闭状态下的晶体结构，Schnell和Chou发表了封闭状态下的核磁共振结构。这些结构与1NYJ模型有很大不同，也没有表现出组氨酸残基之间的螺旋间氢键。除了似乎形成通道门的His和Trp残基外，新结构都有一个由靠近N端的Val残基环形成的狭窄。这被认为至少在一定程度上形成了选择性过滤器；该区域通道的狭窄可以解释为什么通道可以传输质子，而不是其他阳离子物种。

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.