Department of Chemistry and Biochemistry, University of California, Los Angeles, California, USA.
Department of Chemistry, Texas A&M University, College Station, Texas, USA.
Protein Sci. 2024 Apr;33(4):e4973. doi: 10.1002/pro.4973.
Methods in protein design have made it possible to create large and complex, self-assembling protein cages with diverse applications. These have largely been based on highly symmetric forms exemplified by the Platonic solids. Prospective applications of protein cages would be expanded by strategies for breaking the designed symmetry, for example, so that only one or a few (instead of many) copies of an exterior domain or motif might be displayed on their surfaces. Here we demonstrate a straightforward design approach for creating symmetry-broken protein cages able to display singular copies of outward-facing domains. We modify the subunit of an otherwise symmetric protein cage through fusion to a small inward-facing domain, only one copy of which can be accommodated in the cage interior. Using biochemical methods and native mass spectrometry, we show that co-expression of the original subunit and the modified subunit, which is further fused to an outward-facing anti-GFP DARPin domain, leads to self-assembly of a protein cage presenting just one copy of the DARPin protein on its exterior. This strategy of designed occlusion provides a facile route for creating new types of protein cages with unique properties.
蛋白质设计方法使得创建具有多种应用的大型复杂自组装蛋白质笼成为可能。这些方法主要基于高度对称的形式，例如柏拉图立体。通过打破设计对称性的策略，例如，使表面仅显示一个或几个（而不是许多）外部结构域或基序的拷贝，可以扩展蛋白质笼的潜在应用。在这里，我们展示了一种简单的设计方法，用于创建能够显示向外结构域的单个拷贝的对称破缺蛋白质笼。我们通过融合到一个小的向内结构域来修饰一个原本对称的蛋白质笼的亚基，该结构域只能容纳在笼内的一个拷贝。使用生化方法和天然质谱法，我们表明，原始亚基和修饰亚基的共表达，进一步融合到向外的抗 GFP DARPin 结构域上，导致蛋白质笼的自组装，其外部仅呈现一个 DARPin 蛋白的拷贝。这种设计遮挡策略为创建具有独特性质的新型蛋白质笼提供了一种简便途径。
