Kashiwada Ayumi, Matsuda Kiyomi, Mizuno Toshihisa, Tanaka Toshiki
Department of Applied Molecular Chemistry, College of Industrial Technology, Nihon University, 1-2-1 Izumi-cho, Narashino, Chiba, Japan.
Chemistry. 2008;14(24):7343-50. doi: 10.1002/chem.200701726.
In many viruses, pH-responsive coiled-coil domains in the specific fusion proteins play important roles in membrane fusion and the infection of viruses into host cells. To investigate the relationship between the conformational change of the coiled coil and the fusion process, we have introduced a de novo designed polypeptide as a model system of the coiled-coil domain. This system enables the systematic study of the dynamics of pH-responsive coiled-coil polypeptide-membrane interactions. First, we designed and synthesized pH-responsive isoleucine-zipper triple-stranded coiled-coil polypeptides. Then the relationship between the pH-induced conformational change of the polypeptide and the membrane's interactive properties was studied by physicochemical methods. Structural changes in the designed polypeptides were examined by means of circular dichroism measurements. And finally, the behavior of the membrane fusion was investigated by leakage of liposomal contents, turbidity analysis, dynamic light scattering, and lipid mixing experiments. Our data show that coiled-coil formation under acidic pH conditions enhances polypeptide-induced membrane fusion. The results in this study demonstrate that an artificial membrane fusion system can be constructed on a molecular level by the use of a pH-responsive isoleucine-zipper triple-stranded coiled-coil polypeptide.
在许多病毒中，特定融合蛋白中对pH敏感的卷曲螺旋结构域在膜融合以及病毒感染宿主细胞过程中发挥着重要作用。为了研究卷曲螺旋的构象变化与融合过程之间的关系，我们引入了一种从头设计的多肽作为卷曲螺旋结构域的模型系统。该系统能够对pH响应性卷曲螺旋多肽与膜相互作用的动力学进行系统研究。首先，我们设计并合成了对pH敏感的异亮氨酸拉链三链卷曲螺旋多肽。然后，通过物理化学方法研究了多肽pH诱导的构象变化与膜的相互作用特性之间的关系。通过圆二色性测量检测设计多肽的结构变化。最后，通过脂质体内容物泄漏、浊度分析、动态光散射和脂质混合实验研究膜融合行为。我们的数据表明，酸性pH条件下卷曲螺旋的形成增强了多肽诱导的膜融合。本研究结果表明，利用对pH敏感的异亮氨酸拉链三链卷曲螺旋多肽可以在分子水平上构建人工膜融合系统。
