COMPUTER SIMULATION OF THE INFLUENZA M2 CHANNEL

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。在当代生物体中，跨细胞壁运输电荷的蛋白质结构复杂，因此很难剖析其作用的分子机制。因此，希望有一种小的、具有众所周知的结构基序的蛋白质模型，但又能以更复杂的蛋白质的效率和控制进行操作。这导致了对甲型流感M2蛋白的研究--一种小的、同质四聚体的、电压门控的离子通道，它在脂类双层中自组装，并以高效和选择性的方式运输质子。每个单体由97个氨基酸组成，包含一个跨膜结构域。此外，活性通道已经由仅包含25个氨基酸的子集(包括跨膜区)的合成肽重新构建，而不损失特异性或效率。其氨基酸序列为：SerSerAsp-Pro-Leu-Val-Val-Ala-Ala-Ser-Ile-Ile-Gly-Ile-Leu-His-Leu-Ile-Leu-Trp Ile-Leu-Asp-Arg-Leu。与可能是研究最广泛的质子通道模型的格里米菌素A相比，截短的M2通道的质子传输速度要快1000倍以上。这种简单性和效率的非凡结合使M2不仅成为了解简单多肽如何实现高质子运输效率的优秀模型，而且也成为重新设计简单质子泵的一个有吸引力的潜在目标。虽然已知单个螺旋的高分辨率核磁共振结构，但尚未确定四聚体束的结构。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为5.5(也是组氨酸的pKa)以下，该通道打开并观察到质子传输。因此，有人认为，四个中性组氨酸残基来自门，而打开通道涉及质子化一个(或多个)组氨酸残基。克罗斯和他的同事最近的核磁共振工作表明，色氨酸残基与组氨酸残基接近，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编号为1NYJ)。此外，他们最新的核磁共振结果表明，实际的门可能由两个His-His+氢键对组成。基于这一结果，他们认为，在中性pH下，两个组氨酸残基被质子化，当三个(或四个)组氨酸在较低pH下质子化时，门打开。

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. While a high resolution NMR structure for a single helix is known, the structure of the tetrameric bundle has not been determined. 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 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.