Triggered Reversible Disassembly of an Engineered Protein Nanocage**

Abstract

Protein nanocages play crucial roles in sub- cellular compartmentalization and spatial control in all domains of life and have been used as biomolecular tools for applications in biocatalysis, drug delivery, and bionanotechnology. The ability to control their assembly state under physiological conditions would further expand their practical utility. To gain such control, we introduced a peptide capable of triggering conformational change at a key structural position in the largest known encapsulin nanocompartment. We report the structure of the resulting engineered nanocage and demonstrate its ability to disassemble and reassemble on demand under physiological conditions. We demonstrate its capacity for in vivo encapsulation of proteins of choice while also demonstrating in vitro cargo loading capabilities. Our results represent a functionally robust addition to the nanocage toolbox and a novel approach for controlling protein nanocage disassembly and reassembly under mild conditions.A novel protein nanocage has been developed capable of on- demand reversible disassembly via simple buffer exchanges under mild conditions. Data presented herein also show the nanocage is capable of in vivo and in vitro cargo loading, suggesting a broad range of possible applications in biocatalysis, bionanotechnology, and biomedicine. Additional findings include structure determination and protein design verification via cryo- electron microscopy.