Protein encapsulation in the hollow space of hemocyanin crystals containing a covalently conjugated ligand

Encapsulation of guest molecules into the vacant space of biomacromolecular crystals has been utilized for various purposes including functioning as a protein container to protect against physical stress and structural determination of the guest. Todarodes pacificus hemocyanin (TpHc)is a hollow cylindrical decameric protein complex with an inner space 110 Å in diameter and 160 Å in height. In the crystal, TpHc forms a straw-like bundle and contains one reactive Cys (Cys3246)in the inner domain of each protomer. Here, we conjugated biotin onto Cys3246 of TpHc followed by incubation with streptavidin. The streptavidin was immobilized into the inner space of TpHc due to its interaction with biotin. Moreover, the complex containing TpHc and streptavidin was crystallized under the same conditions used for unmodified TpHc. In order to expand this methodology for a variety of proteins, we conjugated the ligand nitrilotriacetic acid (NTA)chelated to a Ni²⁺ ion (Ni²⁺-NTA)to TpHc. We found that His-tagged green fluorescent protein (GFP)was encapsulated into the Ni²⁺-NTA-conjugated TpHc via the interaction between the His-tag and the Ni²⁺-NTA group. X-ray crystallography demonstrated that the crystal packing of the complex containing TpHc and GFP was identical to that of the unmodified TpHc. Our guest immobilization method is distinct from previous approaches that are dependent on diffusion of the guest into the host crystal. Thus, our findings may accelerate the development of proteinaceous crystal engineering.