Protein assemblies have attracted increasing attention for construction of biohybrid materials. Protein crystals can also be regarded as solid protein assemblies. The present work demonstrates that protein crystals can be employed as porous biomaterials by site-specific modifications of the crystals of recombinant sperm whale myoglobin mutants. The myoglobin crystals of space group P6 provide hexagonal pores consisting of the building blocks of six Mb molecules, which form a pore with a diameter of 40 A. On the basis of the lattice structure of the Mb crystals, we have selected appropriate residues located on the surface of the pores for replacement with cysteine. This enables modification of the pore surface via coupling with maleimide derivatives. We have succeeded in crystallizing the modified Mb mutants, retaining the P6 lattice, and consistently aligning nanosized functional molecules such as fluorescein, eosin, and Ru(bpy)(3) into the hexagonal pores of the Mb crystals. Our strategy for site-specific modification of protein crystal pores is applicable to various protein crystals with porous structures. We believe that modified porous protein crystals will provide attractive candidates for novel solid materials in nanotechnology applications.

Nucleotide oligomerization domain-containing protein 2 (Nod2), an innate immune receptor, recognizes bacterial cell-wall peptidoglycan (PGN), the minimum ligand of which is muramyl dipeptide (MDP). Enzymatic digestion of PGN appears to be important for Nod2 recognition. PGN is degraded by muramidase or glucosamidase through a process that produces two types of glycan sequence; glycans containing GlcNAcβ(1→4)MurNAc or MurNAcβ(1→4)GlcNAc. In this report, a range of disaccharide or tetrasaccharide fragments of each sequence were chemically synthesized, and their activities in stimulating human Nod2 (hNod2) were investigated. The results reveal that hNod2 recognitions is dependent on the glycan sequence, as demonstrated by comparing the activities of glycans with the same peptide moieties. (MurNAcβ(1→4)GlcNAc)(2) -containing structures exhibited stronger activity than those containing (GlcNAcβ(1→4)MurNAc)(2) . The results suggest that differences in the enzymatic degradation process affect the host's immunomodulation process.

Peptidoglycan (PGN) is an essential structural component of the bacterial cell wall conferring cell shape, which can be recognized by host-recognition proteins and receptors as well as bacterial surface proteins. In this work, the PGN partial structures from Enterococcus faecalis that contain a tetrasaccharide and an octasaccharide with a unique heptapeptide were synthesized via an Fmoc-strategy for elongation of the glycan chains. Namely, a 4'-O-Fmoc-protected disaccharide was utilized as the key intermediate in this efficient synthetic pathway for preparing various PGN fragments. Both the tetrasaccharide and octasaccharide with the unique heptapeptide were successfully synthesized for the first time.