Haymet A D, Ward L G, Harding M M
Department of Chemistry, University of Houston, TX 77204-5641, USA.
FEBS Lett. 2001 Mar 2;491(3):285-8. doi: 10.1016/s0014-5793(01)02213-x.
The synthesis, solution conformation and ice-growth inhibition properties of four new analogues of the type I 37-residue winter flounder 'antifreeze' protein are reported. All four analogues contain two extra salt bridges to facilitate comparison of results with previously published data. In two analogues, all four threonine residues in the native polypeptide were mutated to 2-amino butyric acid (an unnatural amino acid) and isoleucine, respectively. The butyric acid analogue was approximately 85% helical at 3 degrees C, modified the shape of ice growth, and exhibited reduced hysteresis compared to the native protein (9% at 4 mM). These results show that the gamma-methyl group of threonine, which is present in the sidechain of 2-amino butyric acid, is not sufficient for activity. The isoleucine analogue, in which the threonine hydroxyl group is replaced by an ethyl group, was 100% helical at 3 degrees C, showed no hysteresis but was able to modify the shape of ice crystal growth. In the third and fourth analogues, mutations of the aspartic acids 1 and 5 to alanine, and asparagines 16 and 27 to leucine in the threonine- and valine-substituted analogues did not affect the helicity of the polypeptides, but removed the ability to inhibit ice growth.
报道了I型37个残基的冬鲽“抗冻”蛋白的四种新类似物的合成、溶液构象和冰生长抑制特性。所有四种类似物都含有两个额外的盐桥，以便于将结果与先前发表的数据进行比较。在两种类似物中，天然多肽中的所有四个苏氨酸残基分别突变为2-氨基丁酸（一种非天然氨基酸）和异亮氨酸。丁酸类似物在3℃时约85%呈螺旋状，改变了冰生长的形状，与天然蛋白相比滞后现象减少（4 mM时为9%）。这些结果表明，2-氨基丁酸侧链中存在的苏氨酸γ-甲基不足以产生活性。异亮氨酸类似物中苏氨酸羟基被乙基取代，在3℃时100%呈螺旋状，没有滞后现象，但能够改变冰晶生长的形状。在第三种和第四种类似物中，苏氨酸和缬氨酸取代类似物中天冬氨酸1和5突变为丙氨酸，天冬酰胺16和27突变为亮氨酸，不影响多肽的螺旋度，但消除了抑制冰生长的能力。
