de Groot Bert L, Frigato Tomaso, Helms Volkhard, Grubmüller Helmut
Theoretical Molecular Biophysics Group, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
J Mol Biol. 2003 Oct 17;333(2):279-93. doi: 10.1016/j.jmb.2003.08.003.
Aquaporins are efficient, yet strictly selective water channels. Remarkably, proton permeation is fully blocked, in contrast to most other water-filled pores which are known to conduct protons well. Blocking of protons by aquaporins is essential to maintain the electrochemical gradient across cellular and subcellular membranes. We studied the mechanism of proton exclusion in aquaporin-1 by multiple non-equilibrium molecular dynamics simulations that also allow proton transfer reactions. From the simulations, an effective free energy profile for the proton motion along the channel was determined with a maximum-likelihood approach. The results indicate that the main barrier is not, as had previously been speculated, caused by the interruption of the hydrogen-bonded water chain, but rather by an electrostatic field centered around the fingerprint Asn-Pro-Ala (NPA) motif. Hydrogen bond interruption only forms a secondary barrier located at the ar/R constriction region. The calculated main barrier height of 25-30 kJ mol(-1) matches the barrier height for the passage of protons across pure lipid bilayers and, therefore, suffices to prevent major leakage of protons through aquaporins. Conventional molecular dynamics simulations additionally showed that negatively charged hydroxide ions are prevented from being trapped within the NPA region by two adjacent electrostatic barriers of opposite polarity.
水通道蛋白是高效但具有严格选择性的水通道。值得注意的是，与大多数已知能很好传导质子的其他充满水的孔道不同，质子渗透被完全阻断。水通道蛋白对质子的阻断对于维持跨细胞膜和亚细胞膜的电化学梯度至关重要。我们通过多种非平衡分子动力学模拟研究了水通道蛋白-1中质子排斥的机制，这些模拟还允许质子转移反应。通过模拟，利用最大似然法确定了质子沿通道运动的有效自由能分布。结果表明，主要障碍并非如先前推测的那样是由氢键连接的水链中断引起的，而是由围绕指纹基序天冬酰胺-脯氨酸-丙氨酸（NPA）的静电场引起的。氢键中断仅形成位于芳香/精氨酸（ar/R）收缩区的次要障碍。计算得出的主要障碍高度为25 - 30 kJ·mol⁻¹，与质子穿过纯脂质双层的障碍高度相匹配，因此足以防止质子通过水通道蛋白发生大量泄漏。传统分子动力学模拟还表明，带负电荷的氢氧根离子被两个相邻的相反极性的静电屏障阻止被困在NPA区域内。
