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.

Photoinduced electron transfer (ET) in native photosynthesis reactions is efficiently achieved by the accumulation of different types of redox cofactors within protein assemblies immobilized in cell membranes. [1][2][3] The precise arrangement of each cofactor in the molecular spaces enables them to retain the long-lived charge-separated state, which promotes multistep reactions in biological systems. To elucidate the mechanism of the biological ET reactions and to develop light energy conversion systems, artificial ET proteins have been constructed using de novo proteins, chemical modification of native cofactors, photocatalytic reaction centers engineered into protein assemblies, and design of synthetic metal complexes immobilized in protein-protein ET systems. [4][5][6][7][8][9][10][11][12] The reported systems have provided insights into control of ET rates in terms of the distance between donors and acceptors, hydrogen-bonding interactions, reorganization energy of cofactors, and other factors. [4][5][6][7][8][9][10][11][12] Control of the dense accumulation of the different redox cofactors observed in natural photosystems required to achieve long-lived charge-separated state has caused difficulties in efforts to duplicate this process using artificial protein systems in solution.[13] Thus, the design of novel protein frameworks that allow construction of a dense array of various cofactors is a worthwhile goal. Protein crystals can be regarded as excellent candidates for the development of artificial ET reaction systems because the crystal lattices are expected to allow different types of cofactors to be arranged in three-dimensional frameworks that mimic the native ET systems. ET reactions in single protein crystals have been investigated for the dependence of long-range ET on the structures and orientations of redox centers within proteins. [14][15][16] Gray et al. constructed photochemically-initiated protein-protein ET reactions in protein crystals containing zinc-substituted cytochrome c peroxidase or ruthenium-modified azurin. [14][15][16] Moreover, protein crystals provide nanosized spaces for the fixation of metal ions, metal complexes, and the diffusion of organic molecules. [17][18][19][20][21][22] For instance, accumulation of metal ions and metal complexes in a protein crystal lattice spaces was accomplished simply by soaking of the crystals in a solution containing their precursors. [17][18][19] Anisotropic diffusion of small molecules in hen egg-white lysozyme (HEWL) crystals has been investigated by experimental and simulation approaches. [21,22] The results suggest that these features are governed by steric repulsion and electrostatic interaction induced by amino acid residues located on the internal surface of the crystal lattices. Thus, if we can precisely arrange donor and acceptor molecules and mediators in protein crystals, it is expected that the novel three-dimensional framework will allow us to achieve a longlived charge-separated state.Herein, we construct an artificial long-lived ph...

Protein crystals generally are stable solid protein assemblies. Certain protein crystals are suitable for use as nanovessels for immobilizing metal complexes. Here we report the preparation of ruthenium carbonyl-incorporated cross-linked hen egg white lysozyme crystals (Ru·CL-HEWL). Ru·CL-HEWL retains a Ru carbonyl moiety that can release CO, although a composite of Ru carbonyl-HEWL dissolved in buffer solution (Ru·HEWL) does not release CO. We found that treatment of cells with Ru·CL-HEWL significantly increased nuclear factor kappa B (NF-κB) activity as a cellular response to CO. These results demonstrate that Ru·CL-HEWL has potential for use as an artificial extracellular scaffold suitable for transport and release of a gas molecule.

Porous protein crystals, which are protein assemblies in the solid state, have been engineered to form catalytic vessels by the incorporation of organometallic complexes. Ruthenium complexes in cross-linked porous hen egg white lysozyme (HEWL) crystals catalyzed the enantioselective hydrogen-transfer reduction of acetophenone derivatives. The crystals accelerated the catalytic reaction and gave different enantiomers based on the crystal form (tetragonal or orthorhombic). This method represents a new approach for the construction of bioinorganic catalysts from protein crystals.

Heterogeneous catalysis of cyano-bridged polynuclear metal complexes was examined for the hydrolysis of toxic organophosphates. The surface acidity of cyano-bridged polynuclear metal complexes strongly effects on the catalytic activity.