Aquaporins (AQPs) are α-helical transmembrane proteins that conduct water through membranes with high selectivity and permeability. For human AQP1, in addition to the functional Asn-Pro-Ala motifs and the aromatic/Arg selectivity filter within the pore, there are several highly conserved residues that form an expansive hydrogen-bonding network. Previous solid-state nuclear magnetic resonance studies and structural conservation analysis have detailed which residues may be involved in this network. We explored this network by mutating the side chains or backbones involved in hydrogen-bonding, generating the following mutants: N127A, V133P, E142A, T187A, R195A, and S196A. The fold and stability of these mutants were assessed with attenuated total reflection Fourier transform infrared spectroscopy coupled with hydrogen/deuterium exchange upon increasing temperature. We found that replacement of any of the chosen residues to alanine leads to either partial instability or outright misfolding at room temperature, with the latter being most pronounced for the N127A, V133P, T187A, and R195A mutants. Deconvolution analysis of the amide I band revealed considerable secondary structure deviations, with some mutants exhibiting new random coil and β sheet structures. We also found that some of these mutations potentially disrupt the oligomerization of human AQP1. BN-PAGE and DLS data provide evidence toward the loss of tetramers within most of the mutants, meanwhile only the S196A mutant retains tetrameric organization. The molecular dynamics simulation of the wild-type, and the N127A, E142A, and T187A mutants show that these mutations result in major rearrangements of intra- and intermonomer hydrogen-bond networks. Overall, we show that specific point mutations that perturb hydrogen-bonding clusters result in severe misfolding in hAQP1 and disruption of its oligomerization. These data provide valuable insight into the structural stability of human aquaporin-1 and have implications toward other members of the AQP family, as these networks are largely conserved among a variety of human and nonmammalian AQP homologs.

中文翻译：

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人水通道蛋白-1 中保守的 H 键网络对于天然折叠和寡聚化是必需的


水通道蛋白 （AQP） 是 α 螺旋跨膜蛋白，可以高选择性和渗透性引导水通过膜。对于人 AQP1，除了功能性 Asn-Pro-Ala 基序和孔内的芳香族/Arg 选择性过滤器外，还有几个高度保守的残基形成一个广阔的氢键网络。以前的固态核磁共振研究和结构守恒分析已经详细说明了该网络可能涉及哪些残基。我们通过突变参与氢键的侧链或骨架来探索这个网络，产生以下突变体：N127A、V133P、E142A、T187A、R195A 和 S196A。通过衰减全反射傅里叶变换红外光谱结合氢/氘交换在温度升高时评估这些突变体的折叠和稳定性。我们发现，将任何选定的残基替换为丙氨酸会导致室温下部分不稳定或完全错误折叠，后者对于 N127A、V133P、T187A 和 R195A 突变体最为明显。酰胺 I 带的反卷积分析揭示了相当大的二级结构偏差，一些突变体表现出新的无规则卷曲和β片结构。我们还发现，其中一些突变可能会破坏人 AQP1 的寡聚化。BN-PAGE 和 DLS 数据为大多数突变体中四聚体的缺失提供了证据，同时只有 S196A 突变体保留了四聚体组织。野生型以及 N127A、E142A 和 T187A 突变体的分子动力学模拟表明，这些突变导致单体内和单体间氢键网络的重大重排。 总体而言，我们表明扰乱氢键簇的特定点突变会导致 hAQP1 的严重错误折叠并破坏其寡聚化。这些数据为人类水通道蛋白-1 的结构稳定性提供了有价值的见解，并对 AQP 家族的其他成员具有影响，因为这些网络在各种人类和非哺乳动物 AQP 同系物中基本保守。