Zhang W, Laursen R A
Department of Chemistry, Boston University, Boston, Massachusetts 02215, USA.
J Biol Chem. 1998 Dec 25;273(52):34806-12. doi: 10.1074/jbc.273.52.34806.
Several analogs of an alanine-rich, alpha-helical type I antifreeze polypeptide from the winter flounder were synthesized and studied to evaluate the role of threonine residues on antifreeze activity. In this series, the four Thr residues in the wild type polypeptide were substituted with from one to four Ser, allo-Thr, or Val residues. Circular dichroism studies determined that these substitutions did not significantly diminish alpha-helicity. Thermal hysteresis data showed that substitution of Thr by Ser resulted in moderate to complete loss of antifreeze activity, depending on the number and position of the substituted Thr residue(s). Replacement by Val, in confirmation of other recent reports, or by allo-Thr had a much less detrimental effect on activity though there were qualitative differences in activity between the mutants and the wild type AFP. Based on these results, we propose that both the methyl and hydroxyl groups of Thr, particularly of the central two Thr residues, Thr13 and Thr24, play key roles in the ice-binding properties of the antifreeze peptide. Specifically, the methyls participate in hydrophobic interactions with ice, which provide the driving force for binding and stability, whereas the hydroxyls and other polar residues control binding specificity and impart additional stability through hydrogen bonding.
合成并研究了来自冬鲽的富含丙氨酸的α-螺旋I型抗冻多肽的几种类似物，以评估苏氨酸残基对抗冻活性的作用。在该系列中，野生型多肽中的四个苏氨酸残基被一到四个丝氨酸、别苏氨酸或缬氨酸残基取代。圆二色性研究确定这些取代不会显著降低α-螺旋性。热滞数据表明，用丝氨酸取代苏氨酸会导致抗冻活性中度至完全丧失，这取决于被取代的苏氨酸残基的数量和位置。正如其他近期报道所证实的，用缬氨酸或别苏氨酸取代对活性的不利影响要小得多，尽管突变体和野生型抗冻蛋白之间在活性上存在质的差异。基于这些结果，我们提出苏氨酸的甲基和羟基，特别是中间的两个苏氨酸残基（苏氨酸13和苏氨酸24）的甲基和羟基，在抗冻肽的冰结合特性中起关键作用。具体而言，甲基参与与冰的疏水相互作用，这为结合和稳定性提供驱动力，而羟基和其他极性残基控制结合特异性并通过氢键赋予额外的稳定性。
