Institute for Protein Design, University of Washington, Seattle, Washington, USA.
Molecular Life Sciences, Technical University of Munich, Munich, Germany.
Protein Sci. 2023 Nov;32(11):e4769. doi: 10.1002/pro.4769.
Targeted intracellular delivery via receptor-mediated endocytosis requires the delivered cargo to escape the endosome to prevent lysosomal degradation. This can in principle be achieved by membrane lysis tightly restricted to endosomal membranes upon internalization to avoid general membrane insertion and lysis. Here, we describe the design of small monomeric proteins with buried histidine containing pH-responsive hydrogen bond networks and membrane permeating amphipathic helices. Of the 30 designs that were experimentally tested, all expressed in Escherichia coli, 13 were monomeric with the expected secondary structure, and 4 designs disrupted artificial liposomes in a pH-dependent manner. Mutational analysis showed that the buried histidine hydrogen bond networks mediate pH-responsiveness and control lysis of model membranes within a very narrow range of pH (6.0-5.5) with almost no lysis occurring at neutral pH. These tightly controlled lytic monomers could help mediate endosomal escape in designed targeted delivery platforms.
通过受体介导的内吞作用进行靶向细胞内递送，需要将递送到的货物从内涵体中逃逸出来，以防止溶酶体降解。这在理论上可以通过严格限制在内涵体内化时对内涵体膜的膜裂解来实现，以避免一般的膜插入和裂解。在这里，我们描述了具有埋藏组氨酸的小分子单体蛋白的设计，该蛋白含有 pH 响应氢键网络和膜穿透的两亲性螺旋。在经过实验测试的 30 个设计中，所有在大肠杆菌中表达的设计都是单体，具有预期的二级结构，并且 4 个设计以 pH 依赖性的方式破坏了人工脂质体。突变分析表明，埋藏的组氨酸氢键网络介导 pH 响应性，并在非常窄的 pH 范围内（6.0-5.5）控制模型膜的裂解，在中性 pH 下几乎没有裂解发生。这些紧密控制的溶解释放单体可以帮助介导设计的靶向递药平台中的内涵体逃逸。
