Functional ferritin-like proteins

Herein are described attempts to enclose zero-valent metal nanoparticles within the cavities of the spherical, multimeric proteins from the ferritin family. Nanometer-scale particles of gold and silver have a range of exciting optical and electronic properties, suggesting applications in biomedical imaging, theraputics and materials design. Interfacing these particles with biological systems, such as ferritin, promises greater control over the chemistry and location of the resulting assemblies. Initial efforts showed that the use of commercially available, wildtype protein did not allow the formation of encapsulated particles by means of an in situ reduction of metal ions. However, it motivated the development of ways of studying the formation and characteristics of protein/metal conjugates. A computer algorithm was applied to design a series of mutations to ferritin proteins that would probe the affects of surface chemistry on the stability of the protein and its ability to encapsulate nanoparticles. It was found that the smaller, ferritin-like protein Dps (DNA binding protein from starved cells) could tolerate up to 120 potentially destabilizing mutations to hydrophobic residues on its interior surface. Subsequent work applied 192 mutations to human H ferritin, removing soft-metal-binding ligands from the exterior and adding metal-binding cysteine residues to the cavity. This extensive mutagenesis scheme allowed the formation of small gold and silver clusters inside the protein. However, crystallography studies suggested that the particle size was limited by slow entry of ions into the cavity. A new approach to the encapsulation problem utilized the multimeric nature of ferritin: by chosing a ferritin with reversible, salt-mediated assembly characteristics, it was possible to make protein/metal conjugates using commerically-available gold particles. Transmission electron microscopy (TEM) confirmed that particles could be efficiently encapsulated, filling the entire cavity of the protein. Additionally, studies of surface plasmon resonance (SPR) absorption and fluorophore quenching suggest a near-native protein stoichiometry and stability assays indicate increased tolerance of physiological salt concentrations. An effective method for encapsulation has provided a starting point for the development of protein-induced structuring schemes