Surface-linked molecular monolayers of an engineered myoglobin: Structure, stability, and function

The maintenance of active conformation and biological function is important for the development of solid substrate immobilized biomacromolecules for material applications. A protein monolayer was obtained on SiO2 substrates by chemically linking a site-directed mutant of sperm whale myoglobin, A126C, which has a unique and reactive cysteine residue on its surface, to a thiol specific functional group on the silane-derivatized substrates. Fourier transform infrared (FTIR) spectroscopy of this protein monolayer suggests that a native-like secondary structure is retained for the immobilized myoglobin. In addition, the immobolized myoglobin retains its ability to bind carbon monoxide. Structural changes in the surface-bound protein were examined under variation of temperature, pH, urea concentration, and ethanol content by UV-vis absorption spectroscopy. The densely packed protein, roughly 60% surface coverage of the substrate, is as resilient to high temperature, low-pH environment, and high concentration of urea as myoglobin solution is at low concentration. Surprisingly, we found that after immobilization, the protein resists ethanol denaturation more efficiently than a diluted solution of 1 mu M myoglobin with ethanol as the cosolvent. Intriguingly, the secondary structure of the immobilized myoglobin was preserved after 30 min incubation at 150 degrees C, as determined by FTIR at room temperature.