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.

Dielectric properties of Bauhinia monandra and Concanavalin A lectin monolayers, part I

“…The molecular organization of a spread protein after its conformational stabilization would necessarily involve exposure of its amino acids towards the aqueous phase. A protein under such conditions normally maintains its secondary structure, but loses its original 3D conformation [10]. Different protein monolayers were found to be stable and preserve their native conformation after spreading [11][12][13].…”

Polarized XAS on vectorially oriented single monolayers of cytochrome c

“…While other research groups have focused on measurements of the functional aspects of such (or closely related e.g. Langmuir-Blodgett) monolayer systems that were not structurally characterized (Song et al, 1993;Cullinson et al, 1994;Owaku et al, 1995;Jiang et al, 1996;Guo et al, 1996), we have focused instead on developing the physical techniques essential to determining the key structural features of the proteins within such vectorially oriented single monolayers. To date, this work has included resonance x-ray diffraction (Pachence et al, 1989), optical linear dichroism (Pachence et al, 1990), and x-ray interferometry/holography (Blasie et al, 1992;Chupa et ai., 1994;Edwards et al, 1997 andEdwards et al, 1998).…”

Synthesis and Self‐Assembly of Organoclay‐Wrapped Biomolecules

“…[1][2][3] Although proteins excel in functional specificity, their structural and chemical sensitivity to ambient conditions can seriously compromise the use and integration of such macromolecules in diverse applications. Although there are numerous reports on the enhanced thermal and chemical stability of proteins by immobilization on surfaces [4][5][6][7][8] or within matrices such as amorphous gels [9][10][11] and layered solids, [12,13] the wrapping of individual protein/enzyme molecules with inorganic materials to produce functionally isolated hybrid nanoparticles has not been reported. Herein we describe investigations that strongly suggest that individual molecules of met-myoglobin (Mb), haemoglobin (Hb) or glucose oxidase (GOx) can be wrapped with an ultrathin shell of an aminopropyl-functionalized magnesium (organo)phyllosilicate to produce aqueous dispersions of discrete protein-inorganic nanoparticles.…”

“…The organoclay was prepared by chemical synthesis [14,15] (see Experimental Section) and consisted of a highly disordered talclike 2:1 trioctahedral smectite structure with a central brucite sheet of octahedrally coordinated MgO 4 . Protonation of the amino groups by dispersion of the clay in water resulted in exfoliation and partial disintegration of the organoclay layers into cationic oligomers that were fractionated by gel chromatography to produce stable transparent sols that were subsequently added to protein/enzyme solutions.…”