Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0624, United States.
Department of Biomedical Engineering, University of Michigan Medical School, Ann Arbor, Michigan 48109-1382, United States.
Biomacromolecules. 2023 Mar 13;24(3):1388-1399. doi: 10.1021/acs.biomac.2c01424. Epub 2023 Feb 16.
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 . 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.
包被蛋白是微生物蛋白纳米笼，能够高效地自组装并包裹酶类货物。由于其具有高耐热性、抗蛋白酶性和稳健的异源表达等优良特性，包被蛋白已成为医学、催化和纳米技术等领域应用的热门生物工程工具。在高温和低 pH 等极端物理化学条件下具有抗性，是许多生物技术应用的理想特性。然而，目前还没有针对耐酸包被蛋白进行系统搜索，而 pH 对包被蛋白壳的影响也尚未得到彻底研究。在这里，我们报告了一种新鉴定的耐酸细菌中的包被蛋白纳米笼。我们使用透射电子显微镜、动态光散射和蛋白水解测定法，证明了其极端的耐酸性和对蛋白酶的抵抗力。我们使用冷冻电子显微镜对新型纳米笼进行了结构表征，揭示了其独特的五重孔，在中性 pH 下呈现明显的“关闭”和“打开”状态，但在强酸性条件下仅呈现单一的“关闭”状态。此外，“打开”状态显示了迄今为止报道的包被蛋白壳中最大的孔。我们还展示了非天然蛋白质的包裹能力，并探讨了外部 pH 对内部货物的影响。我们的研究结果扩展了包被蛋白纳米笼的生物技术应用范围，使其有望在强酸性条件下使用，并强调了 pH 响应的包被蛋白孔动力学。
