Reversible Assembly of Virus-Like Particles (VLPs) into Higher-Order Structures Controlled by Oxidation and Reduction of Linker Protein.

Controlled assembly of nanoparticles into higher-order structures is of great interest, because such materials have the potential to exhibit collective properties distinct from those of individual particles. Introducing the capability for reversible assembly and disassembly behavior in response to an environmental stimulus further enables the development of stimulus-responsive smart array materials. In this study, we demonstrate the reversible assembly of protein building blocks into higher-order structures mediated by the oxidation and reduction of thiol groups incorporated into a linker protein. As the building block, we utilized the P22 virus-like particle (VLP), a 60 nm cage-like protein derived from bacteriophage P22 that can encapsulate a variety of cargo molecules. The linker was derived from the decoration (Dec) protein, a homotrimeric protein that binds to symmetry-specific sites on the exterior surface of the matured form of the P22 capsid. We engineered a Dec mutant, DecS134C, by replacing the C-terminal amino acid of Dec with cysteine, enabling the formation of a "back-to-back" dimer (Dec-S-S-Dec) through disulfide bond formation that functions as a ditopic linker. Because each P22 VLP presents 80 Dec binding sites, Dec-S-S-Dec dimers cross-link P22 VLPs to form higher-order three-dimensional arrays. The disulfide bonds in the linkers are cleaved and reformed upon reduction and oxidation, respectively, leading to the reversible disassembly and reassembly of higher-order VLP arrays controlled by redox conditions. Under optimal conditions, disassembly and reassembly were completed within 30 and 5 min, respectively. This study demonstrates a redox-controlled strategy for the reversible assembly and disassembly of VLP-based materials and provides a versatile platform for constructing stimulus-responsive protein array materials.