The Nagayama Protein Array project attempted to establish a universal technology for fabricating two-dimensional protein arrays in the form of crystalline or amorphous films having specifically desired molecular orientations and alignments.

Supramolecules: A new architecture of nature involving carbohydrate gluing (polysacchride-lectin like binding) was established for the supramolecular assembly of huge proteins comprising about 200 protein subunits, giant hemoglobin from annelid. Covalently bonded dimers and trimers of ribonuclease H were synthesized through genetic fusion by gene engineering and chemical fusion by trifunctionals, which connect three monomers through surface cysteins. Liposomes were used as templates to assemble ferritin molecules by introducing lock-and-key interactions: avidin-biotin and lectin-polysaccharide. Virus-like protein vesicles were organized by tailoring specific (lock-and-key) and non-specific (electrostatic) interactions. Particle and Protein Assembly A novel fabrication principle applicable to colloidal particles including proteins for their two-dimensional self-assembly was found. Particles which are mobile in thin liquid films possessing a thickness comparable to the particles were assembled and hexagonally packed into crystalline arrays. The continuous growth of a monolayer of particle arrays with crystalline order was obtained with a simple apparatus by only vertically dipping and withdrawing a wettable glass to and from a colloid suspension. Two-dimensional Protein Crystals Thick liquid film systems for preparing protein 2D arrays were developed by employing the secondary minimum induced by the electric potential near the charged surface. Hexagonal, tetragonal and oblique lattice ferritin 2D crystals were prepared on thin liquid films developed on a glucose solution by tailoring interprotein interactions through introducing mutations on protein surfaces. Characterization and Simulation of Arrays A color ellipsocope was developed to monitor the in situ growth of protein arrays. Various VIDEO microscope techniques were developed to monitor film formation, particle-array formation and protein-array formation. The function of ferritin, ferroxidase activity, in 2D crystals made on mercury and transferred to a carbon film in the dry form was monitored by a new method: electron microscopic assay of the function for a single protein. Crystal stability by electrostatic interactions were legitimated by computer simulation for real 3D crystals of three proteins: insulin, basic pancreatic trypsin inhibitor and ferritin. A poker-chip model used to study the physical aspects of molecular-assembly and molecular interactions was intensively developed.