Surface immobilization of engineered hemeproteins: Orientation, stability, structure, and function

Biologically derived macromolecules have drawn great interests for their potentials in biotechnology and material applications. Hemeproteins, including myoglobin, cytochrome b;Self-assembled protein monolayers were formed on SiO;A unique cysteine residue was introduced onto the surface of protein molecules at different positions by site-directed mutagenesis. Differential heme orientations were achieved through the choice of the attachment site, which demonstrated a remarkable approach to manipulate molecular orientation in protein monolayers. Secondary structure of the molecular monolayer proteins was investigated by Fourier transform infrared spectroscopy. In addition, structure changes of the surface-bound proteins were examined by UV-Vis absorption spectroscopy of the heme chromophore under the variation of temperature, pH, urea concentration and ethanol content. It was concluded that protein secondary structure is highly maintained even after thermal treatment and that surface-immobilized proteins have similar stability as solution proteins. Maintenance of the biological functions of the substrate-bound myoglobin was indicated by its ability of binding to different ligands.;In summary, the controllable protein orientation, the high protein stability and the maintenance of biological activity make this surface-immobilized protein monolayer an excellent system for material applications of hemeproteins.