Within the cell, chemical reactions are often confined and organized through a modular architecture. This facilitates the targeted localization of molecular species and their efficient translocation to subsequent sites. Here we present a cell-free nanoscale model that exploits compartmentalization strategies to carry out regulated protein unfolding and degradation. Our synthetic model comprises two connected DNA origami nanocompartments (each measuring 25 nm × 41 nm × 53 nm): one containing the protein unfolding machine, p97, and the other housing the protease chymotrypsin. We achieve the unidirectional immobilization of p97 within the first compartment, establishing a gateway mechanism that controls substrate recruitment, translocation and processing within the second compartment. Our data show that, whereas spatial confinement increases the rate of the individual reactions by up to tenfold, the physical connection of the compartmentalized enzymes into a chimera efficiently couples the two reactions and reduces off-target proteolysis by almost sixfold. Hence, our modular approach may serve as a blueprint for engineering artificial nanofactories with reshaped catalytic performance and functionalities beyond those observed in natural systems.

在单元内，化学反应通常通过模块化架构进行限制和组织。这有助于分子种类的靶向定位及其向后续位点的有效转位。在这里，我们提出了一个无细胞纳米模型，该模型利用区室化策略来执行受调节的蛋白质展开和降解。我们的合成模型包括两个连接的 DNA 折纸纳米隔室（每个尺寸为 25 nm × 41 nm × 53 nm）：一个包含蛋白质展开机 p97，另一个包含蛋白酶胰凝乳蛋白酶。我们在第一隔室内实现了 p97 的单向固定，建立了一个控制第二隔室内底物募集、易位和加工的门户机制。我们的数据表明，虽然空间限制将单个反应的速率提高了十倍，但区室化酶的物理连接成嵌合体有效地耦合了两个反应，并将脱靶蛋白水解减少了近六倍。因此，我们的模块化方法可以作为工程人造纳米工厂的蓝图，其重塑的催化性能和功能超出了在自然系统中观察到的。