Department of Applied Molecular Chemistry, Graduate School of Industrial Technology, Nihon University, Narashino, Chiba, Japan.
Langmuir. 2011 Feb 15;27(4):1403-8. doi: 10.1021/la103908u. Epub 2010 Nov 8.
The coiled coil trimer structure is a common motif observed in membrane fusion processes of specific fusion proteins such as the hemagglutinin glycoprotein. The HA2 subunit in the hemagglutinin changes its conformation or geometry to be favorable to membrane fusion in response to endosomal weakly acidic pH. This pH responsiveness is indispensable to an artificial polypeptide-triggered delivery system as well as the membrane fusion reaction in biology. In this study, we have constructed an AAB-type coiled coil heteroassembled system that is sensitive to weakly acidic pH. The heterotrimer is formed from two kinds of polypeptides containing an Ala or a Trp residue at a hydrophobic a position, and it was observed that the Glu residue at the other a position induced an acidic pH-dependent conformational change. On the basis of this pH-responsive coiled coil heteroassembled system, a boronic acid coupled working polypeptide for the combination of an intervesicular complex with a sugarlike compound on the surface of the target liposome, and a supporting polypeptide for the construction of a pH-responsive heterotrimer with the working polypeptide were designed and synthesized. The process of membrane fusion was characterized by lipid-mixing, inner-leaflet lipid-mixing, and content-mixing assays. The target selective vesicle fusion is clearly observed at a weakly acidic pH, where the working polypeptides form a heterotrimeric coiled coil with the supporting polypeptides in a 1:2 binding stoichiometry and the surfaces between pilot and target vesicles come into close proximity to each other.
卷曲螺旋三聚体结构是一种常见的模式，存在于特定融合蛋白的膜融合过程中，如血凝素糖蛋白。血凝素中的 HA2 亚基在响应内体弱酸性 pH 时改变其构象或几何形状，有利于膜融合。这种对 pH 的响应对于人工多肽触发的递药系统以及生物学中的膜融合反应都是必不可少的。在这项研究中，我们构建了一种对弱酸性 pH 敏感的 AAB 型卷曲螺旋杂组装系统。杂三聚体由两种多肽组成，其中一种多肽在疏水性 a 位含有丙氨酸或色氨酸残基，另一种多肽在另一个 a 位含有谷氨酸残基，该残基诱导酸性 pH 依赖性构象变化。基于这种对 pH 敏感的卷曲螺旋杂组装系统，我们设计并合成了一种硼酸盐偶联的工作多肽，用于结合含有目标脂质体表面糖样化合物的囊泡内复合物，以及一种支持多肽，用于与工作多肽构建 pH 响应的杂三聚体。通过脂质混合、内层脂质混合和内容物混合测定来表征膜融合过程。在弱酸性 pH 下，明显观察到目标选择性囊泡融合，其中工作多肽与支持多肽以 1:2 的结合计量比形成杂三聚体卷曲螺旋，并且先导和目标囊泡之间的表面彼此紧密接近。
