Characterizing the Dynamic Disassembly/Reassembly Mechanisms of Encapsulin Protein Nanocages

Encapsulins, self-assembling icosahedral protein nanocages derived from prokaryotes, represent a versatile set of tools for nanobiotechnology. However, a comprehensive understanding of the mechanisms underlying encapsulin self-assembly, disassembly, and reassembly is lacking. Here, we characterize the disassembly/reassembly properties of three encapsulin nanocages that possess different structural architectures: T = 1 (24 nm), T = 3 (32 nm), and T = 4 (42 nm). Using spectroscopic techniques and electron microscopy, encapsulin architectures were found to exhibit varying sensitivities to the denaturant guanidine hydrochloride (GuHCl), extreme pH, and elevated temperature. While all three encapsulins showed the capacity to reassemble following GuHCl-induced disassembly (within 75 min), only the smallest T = 1 nanocage reassembled after disassembly in basic pH (within 15 min). Furthermore, atomic force microscopy revealed that all encapsulins showed a significant loss of structural integrity after undergoing sequential disassembly/reassembly steps. These findings provide insights into encapsulins' disassembly/reassembly dynamics, thus informing their future design, modification, and application.

Automated summary

Encapsulins, self-assembling icosahedral protein nanocages derived from prokaryotes, show varying sensitivities to denaturant, extreme pH, and elevated temperature, and exhibit different reassembly capabilities after disassembly. While all three encapsulins were able to reassemble following GuHCl-induced disassembly, only the smallest T = 1 nanocage could reassemble after disassembly in basic pH. Atomic force microscopy revealed a significant loss of structural integrity in all encapsulins after sequential disassembly/reassembly steps. These findings provide insights into the dynamics of encapsulins' disassembly/reassembly, informing their future design, modification, and application.