Yip C M, DeFelippis M R, Frank B H, Brader M L, Ward M D
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, USA.
Biophys J. 1998 Sep;75(3):1172-9. doi: 10.1016/S0006-3495(98)74036-6.
Although x-ray crystal structures exist for many forms of insulin, the hormone involved in glucose metabolism and used in the treatment of diabetes, x-ray structural characterization of therapeutically important long-acting crystalline ultralente insulin forms has been elusive because of small crystal size and poor diffraction characteristics. We describe tapping-mode atomic force microscopy (TMAFM) studies, performed directly in crystallization liquor, of ultralente crystals prepared from bovine, human, and porcine insulins. Lattice images obtained from direct imaging of crystal planes are consistent with R3 space group symmetry for each insulin type, but the morphology of the human and porcine crystals observed by AFM differs substantially from that of the bovine insulin crystals. Human and porcine ultralente crystals exhibited large, molecularly flat (001) faces consisting of hexagonal arrays of close packed hexamers. In contrast, bovine ultralente crystals predominantly exhibited faces with cylindrical features assignable to close-packed stacks of insulin hexamers laying in-plane, consistent with the packing motif of the (010) and (011) planes. This behavior is attributed to a twofold increase in the hydrophobic character of the upper and lower surfaces of the donut-shaped insulin hexamer in bovine insulin compared to its human and porcine counterparts that results from minor sequence differences between these insulins. The increased hydrophobicity of these surfaces can promote hexamer-hexamer stacking in precrystalline aggregates or enhance attachment of single hexamers along the c axis at the crystal surface during crystal growth. Both events lead to enhanced growth of ¿hk0¿ planes instead of (001). The insulin hexamers on the (010) and (110) faces are exposed "edge-on" to the aqueous medium, such that solvent access to the center of the hexamer and to solvent channels is reduced compared to the (001) surface, consistent with the slower dissolution and reputed unique basal activity of bovine ultralente insulin. These observations demonstrate that subtle variations in amino acid sequence can dramatically affect the interfacial structure of crystalline proteins.
尽管存在多种形式胰岛素的X射线晶体结构，胰岛素是参与葡萄糖代谢并用于治疗糖尿病的激素，但由于晶体尺寸小和衍射特性差，具有治疗重要性的长效结晶超长效胰岛素形式的X射线结构表征一直难以实现。我们描述了在结晶液中直接进行的轻敲模式原子力显微镜（TMAFM）研究，该研究针对由牛、人及猪胰岛素制备的超长效晶体。从晶体平面直接成像获得的晶格图像与每种胰岛素类型的R3空间群对称性一致，但通过原子力显微镜观察到的人胰岛素和猪胰岛素晶体的形态与牛胰岛素晶体的形态有很大不同。人胰岛素和猪胰岛素超长效晶体呈现出大的、分子平坦的（001）面，该面由紧密堆积的六聚体的六边形阵列组成。相比之下，牛胰岛素超长效晶体主要呈现出具有圆柱形特征的面，这些面可归因于平面内胰岛素六聚体的紧密堆积堆叠，这与（010）和（011）平面的堆积基序一致。这种行为归因于与人和猪胰岛素对应物相比，牛胰岛素中甜甜圈形胰岛素六聚体上下表面的疏水特性增加了两倍，这是由这些胰岛素之间的微小序列差异导致的。这些表面增加的疏水性可促进预结晶聚集体中六聚体 - 六聚体的堆叠，或在晶体生长过程中增强单个六聚体沿晶体表面c轴的附着。这两个事件都会导致¿hk0¿平面而不是（001）平面的生长增强。（010）和（110）面上的胰岛素六聚体“边缘”暴露于水性介质中，因此与（001）表面相比，溶剂进入六聚体中心和溶剂通道的机会减少，这与牛胰岛素超长效胰岛素较慢的溶解和所谓独特的基础活性一致。这些观察结果表明氨基酸序列的细微变化可显著影响结晶蛋白质的界面结构。
