Surface-Linked Molecular Monolayers of an Engineered Myoglobin: Structure, Stability, and Function

Abstract

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 μM myoglobin with ethanol as the cosolvent. Intriguingly, the secondary structure of the immobilized myoglobin was preserved after 30 min incubation at 150 °C, as determined by FTIR at room temperature.