IFLYSIB, UNLP-CONICET, Calle 59, 789, B1900BTE, La Plata, Argentina.
J Mol Recognit. 2011 Jul-Aug;24(4):724-32. doi: 10.1002/jmr.1130.
Antifreeze proteins (AFPs) inhibit ice growth at sub-zero temperatures. The prototypical type-III AFPs have been extensively studied, notably by X-ray crystallography, solid-state and solution NMR, and mutagenesis, leading to the identification of a compound ice-binding surface (IBS) composed of two adjacent ice-binding sections, each which binds to particular lattice planes of ice crystals, poisoning their growth. This surface, including many hydrophobic and some hydrophilic residues, has been extensively used to model the interaction of AFP with ice. Experimentally observed water molecules facing the IBS have been used in an attempt to validate these models. However, these trials have been hindered by the limited capability of X-ray crystallography to reliably identify all water molecules of the hydration layer. Due to the strong diffraction signal from both the oxygen and deuterium atoms, neutron diffraction provides a more effective way to determine the water molecule positions (as D(2) O). Here we report the successful structure determination at 293 K of fully perdeuterated type-III AFP by joint X-ray and neutron diffraction providing a very detailed description of the protein and its solvent structure. X-ray data were collected to a resolution of 1.05 Å, and neutron Laue data to a resolution of 1.85 Å with a "radically small" crystal volume of 0.13 mm(3). The identification of a tetrahedral water cluster in nuclear scattering density maps has allowed the reconstruction of the IBS-bound ice crystal primary prismatic face. Analysis of the interactions between the IBS and the bound ice crystal primary prismatic face indicates the role of the hydrophobic residues, which are found to bind inside the holes of the ice surface, thus explaining the specificity of AFPs for ice versus water.
抗冻蛋白（AFPs）在低温下抑制冰的生长。典型的 III 型 AFP 已经被广泛研究，特别是通过 X 射线晶体学、固态和溶液 NMR 以及诱变，从而确定了一个由两个相邻的冰结合部分组成的复合冰结合表面（IBS），每个部分都与冰晶的特定晶格平面结合，从而毒害它们的生长。这个表面包括许多疏水性和一些亲水性残基，已经被广泛用于模拟 AFP 与冰的相互作用。实验观察到的面对 IBS 的水分子被用于尝试验证这些模型。然而，这些尝试受到 X 射线晶体学可靠地识别水合层中所有水分子的能力的限制。由于氧和氘原子的强衍射信号，中子衍射提供了一种更有效的方法来确定水分子的位置（作为 D（2）O）。在这里，我们报告了通过 X 射线和中子衍射联合成功地确定了完全氘代 III 型 AFP 的结构，在 293 K 下，这为蛋白质及其溶剂结构提供了非常详细的描述。X 射线数据的收集分辨率为 1.05 Å，中子劳埃数据的分辨率为 1.85 Å，晶体体积非常小，只有 0.13 mm（3）。在核散射密度图中鉴定出一个四面体水分子簇，使得重建 IBS 结合的冰晶初级棱柱面成为可能。对 IBS 与结合冰晶初级棱柱面之间相互作用的分析表明了疏水性残基的作用，这些残基被发现结合在冰表面的孔内，从而解释了 AFP 对冰与水的特异性。
