Crystalline porous materials have been investigated for development of important applications in molecular storage, separations, and catalysis. The potential of protein crystals is increasing as they become better understood. Protein crystals have been regarded as porous materials because they present highly ordered 3D arrangements of protein molecules with high porosity and wide range of pore sizes. However, it remains difficult to functionalize protein crystals in living cells. Here, we report that polyhedra, a natural crystalline protein assembly of polyhedrin monomer (PhM) produced in insect cells infected by cypovirus, can be engineered to extend porous networks by deleting selected amino acid residues located on the intermolecular contact region of PhM. The adsorption rates and quantities of fluorescent dyes stored within the mutant crystals are increased relative to those of the wild-type polyhedra crystal (WTPhC) under both in vitro and in vivo conditions. These results provide a strategy for designing self-assembled protein materials with applications in molecular recognition and storage of exogenous substances in living cell as well as an entry point for development of bioorthogonal chemistry and in vivo crystal structure analysis.

A crystalline protein assembly of cypovirus polyhedra was engineered to develop a carbon monoxide (CO) releasing extracellular scaffold by immobilizing ruthenium carbonyls. The molecular design includes introduction of a hexahistidine tag to the C-terminus and provides immobilization of about 2-fold more Ru carbonyls per protein monomer and effectively releases three times more CO for activation of nuclear factor kappa B (NF-κB) in living cells relative to wild-type polyhedra with Ru carbonyls.

Cross-linked hen egg white lysozyme crystals (CL-HEWL) have been employed as supports to construct heterogeneous catalysts for photocatalytic hydrogen (H2) evolution, where rose bengal (RB) and Pt nanoparticles (PtNPs) acted as a photosensitizer and H2-evolution catalysts, respectively. Single-crystal X-ray structure analyses of the CL-HEWL immobilizing a precursor for PtNPs suggested that a coordination site of the precursor locates in immediate proximity to potential adsorption sites for RB. The accumulation of the components facilitated photo-induced electron transfer, resulting in efficient H2 evolution. These results suggest that porous protein crystals are promising platforms to periodically and rationally accumulate catalytic components by using molecular interactions.

Photoactivatable CO releasing protein crystals were developed by immobilization of Mn carbonyl complexes in polyhedral crystals, which are spontaneously formed in insect cells. The photoactivatable CO release from the engineered protein crystals activates nuclear factor kappa B (NF-κB) upon stimulation by visible light irradiation with suppression of cytotoxicity of the Mn complex.