Structural basis for the superior activity of the large isoform of snow flea antifreeze protein.
Structural basis for the superior activity of the large isoform of snow flea antifreeze protein.
复制标题
雪蚤抗冻蛋白大亚型的卓越活性的结构基础。
DOI：
10.1021/bi901929n
复制
发表时间：
2010
期刊：
影响因子：
2.9
通讯作者：
P. Davies
中科院分区：
文献类型：
--
作者：
Yee;Feng;L. Graham;Yeliz Celik;I. Braslavsky;P. Davies
雪蚤（Hypogastrum harveyi）在零度以下的温度下可以通过富含甘氨酸的抗冻蛋白（AFP）来防止冻结，该蛋白与冰晶种子结合并防止它们长大。这种AFP是活性亢进的，并且包含两种同种型[Graham，L.一、和Davies，P. L.（2005）Science 310，461]。较大的同种型（15.7 kDa）的活性比较小的同种型（6.5 kDa）高几倍，尽管其丰度要低得多。为了建立这种活性差异的分子基础，我们确定了大同种型的序列。这两种亚型的一级序列惊人地不同。然而，两者都含有三肽重复序列和转向基序，使我们能够建立一个三维模型的基础上的六聚脯氨酸螺旋结构的小亚型的大亚型。我们的模型包含13个聚脯氨酸II型螺旋连接的脯氨酸含环堆叠成两个平面面向彼此反平行。该结构是严格的两亲性，一边是亲水表面，另一边是疏水的，推定的冰结合表面。推定的冰结合位点的面积约为小同种型的两倍，这为与其他例子一致的活性差异提供了解释。用绿色荧光蛋白标记重组AFP，观察其与冰的多个面，尤其是基面的结合。这一发现支持AFP的高活性和基底面结合之间的相关性，首次观察到云杉芽虫AFP。
The snow flea (Hypogastrum harveyi) is protected from freezing at sub-zero temperatures by a glycine-rich antifreeze protein (AFP) that binds to seed ice crystals and prevents them from growing larger. This AFP is hyperactive and comprises two isoforms [Graham, L. A., and Davies, P. L. (2005) Science 310, 461]. The larger isoform (15.7 kDa) exhibits several-fold higher activity than the smaller isoform (6.5 kDa), although it is considerably less abundant. To establish the molecular basis for this difference in activity, we determined the sequence of the large isoform. The primary sequences of these two isoforms are surprisingly divergent. However, both contain tripeptide repeats and turn motifs that enabled us to build a three-dimensional model of the large isoform based upon the six-polyproline helix structure of the small isoform. Our model contains 13 polyproline type II helices connected by proline-containing loops stacked into two flat sheets oriented antiparallel to one another. The structure is strictly amphipathic, with a hydrophilic surface on one side and a hydrophobic, putative ice-binding surface on the other. The putative ice-binding site is approximately twice as large in area as that of the small isoform, providing an explanation for the difference in activity that is consistent with other examples noted. By tagging the recombinant AFP with green fluorescent protein, we observed its binding to multiple planes of ice, especially the basal plane. This finding supports the correlation between AFP hyperactivity and basal plane binding first observed with spruce budworm AFP.