Protein cages are a common architectural motif used by living organisms to compartmentalize and control biochemical reactions. While engineered protein cages have recently been featured in the construction of nanoreactors and synthetic organelles, relatively little is known about the underlying molecular parameters that govern cage stability and molecular flux through their pores. In this work, we systematically designed a 24-member library of protein cage variants based on the T. maritima encapsulin, each featuring pores of different size and charge. Twelve encapsulin pore variants were successfully assembled and purified, including eight designs with exceptional and prolonged thermal stability. While pores lined with negatively charged residues resulted in more robust assemblies than their corresponding positively charged variants, we were able to form stable assemblies covering a full range of pore sizes and charges, as observed in seven new cryo-EM structures of pore variants elucidated at resolutions between 2.5-3.6 Å. Alongside these structures, molecular dynamics simulations and stopped flow kinetics experiments reveal the importance of considering both pore size and surface charge, together with flexibility and rate determining steps, when designing protein cages for controlling molecular flux.

中文翻译：

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孔结构控制工程蛋白质笼中的稳定性和分子通量

蛋白质笼是生物体用来区分和控制生化反应的常见建筑主题。虽然最近设计的蛋白质笼已在纳米反应器和合成细胞器的构建中出现，但对控制笼稳定性和通过其孔的分子通量的潜在分子参数知之甚少。在这项工作中，我们系统地设计了一个基于T. maritima的 24 个成员的蛋白质笼变体库encapsulin，每个都有不同大小和电荷的孔。成功组装和纯化了 12 种封装蛋白孔变体，其中包括 8 种具有出色且持久的热稳定性的设计。虽然内衬带负电残基的孔比其相应的带正电变体产生更强大的组装，但我们能够形成覆盖全范围孔径和电荷的稳定组装，正如在七个新的孔变体的冷冻电镜结构中所观察到的那样分辨率在 2.5-3.6 Å 之间。除了这些结构外，分子动力学模拟和停流动力学实验揭示了在设计用于控制分子通量的蛋白质笼时考虑孔径和表面电荷以及灵活性和速率确定步骤的重要性。