Exploring the Extreme Acid Tolerance of a Dynamic Protein Nanocage

Encapsulins
are microbial protein nanocages capable of efficient
self-assembly and cargo enzyme encapsulation. Due to their favorable
properties, including high thermostability, protease resistance, and
robust heterologous expression, encapsulins have become popular bioengineering
tools for applications in medicine, catalysis, and nanotechnology.
Resistance against physicochemical extremes like high temperature
and low pH is a highly desirable feature for many biotechnological
applications. However, no systematic search for acid-stable encapsulins
has been carried out, while the influence of pH on encapsulin shells
has so far not been thoroughly explored. Here, we report on a newly
identified encapsulin nanocage from the acid-tolerant bacterium Acidipropionibacterium acidipropionici. Using transmission
electron microscopy, dynamic light scattering, and proteolytic assays,
we demonstrate its extreme acid tolerance and resilience against proteases.
We structurally characterize the novel nanocage using cryo-electron
microscopy, revealing a dynamic five-fold pore that displays distinct
“closed” and “open” states at neutral
pH but only a singular “closed” state under strongly
acidic conditions. Further, the “open” state exhibits
the largest pore in an encapsulin shell reported to date. Non-native
protein encapsulation capabilities are demonstrated, and the influence
of external pH on internalized cargo is explored. Our results expand
the biotechnological application range of encapsulin nanocages toward
potential uses under strongly acidic conditions and highlight pH-responsive
encapsulin pore dynamics.

封装蛋白纳米笼（Encapsulin）是一类源自微生物的蛋白质纳米笼，具备高效自组装能力与酶负载包裹功能。凭借高耐热性、抗蛋白酶水解特性及稳定的异源表达能力等优异性能，封装蛋白纳米笼已成为医药、催化与纳米技术领域备受青睐的生物工程工具。耐受高温、低pH等极端理化条件的特性，是众多生物技术应用中极具价值的性能。然而目前尚未开展针对耐酸性封装蛋白纳米笼的系统性筛选研究，且pH对封装蛋白纳米笼壳层的影响也未得到充分探究。本研究报道了一株来自耐酸细菌酸丙酸杆菌（Acidipropionibacterium acidipropionici）的新型封装蛋白纳米笼。通过透射电子显微镜、动态光散射与蛋白酶水解实验，本研究证实了该纳米笼极强的耐酸性与抗蛋白酶水解能力。借助冷冻电子显微镜对该新型纳米笼进行结构解析，发现其存在一个动态五重对称孔道：在中性pH条件下可呈现"开放"与"闭合"两种截然不同的状态，而在强酸性条件下仅存在单一的"闭合"状态。此外，该纳米笼"开放"状态下的孔道尺寸为目前已报道的封装蛋白纳米笼中最大。本研究还证实了该纳米笼对外源蛋白质的包裹能力，并探究了外部pH对内部负载物的影响。本研究结果拓展了封装蛋白纳米笼的生物技术应用场景，使其可适用于强酸性条件下的潜在应用，并凸显了封装蛋白纳米笼孔道的pH响应动态特性。