Abstract
The encapsulation of biomolecules in solid materials that retain the native properties of the molecule is a desired feature for the development of biosensors and biocatalysts. In the current study, protein entrapment in silica-based materials is explored using the sol-gel technique. This work surveys the effects of silica confinement on the structure of several model polypeptides, including apomyoglobin, copper-zinc superoxide dismutase, polyglutamine, polylysine, and type I antifreeze protein. Changes in the secondary structure of each protein following encapsulation are monitored by circular dichroism spectroscopy. In many cases, silica confinement reduces the fraction of properly-folded protein relative to solution, but addition of a secondary solute or modification of the silica surface leads to an increase in structure. Refinement of the glass surface by addition of a monosubstituted alkoxysilane during sol-gel processing is shown to be a valuable tool for testing the effects of surface chemistry on protein structure. Because silica entrapment prevents protein aggregation by isolating individual protein molecules in the pores of the glass material, one may monitor aggregation-prone polypeptides under solvent conditions that are prohibited in solution, as demonstrated with polyglutamine and a disease-related variant of superoxide dismutase.
Highlights
The characterization of biomolecules in dilute solution is useful for determining the properties of the purified molecule, this approach fails to account for excluded volume and hydration effects that occur in concentrated, non-ideal solutions [1]
The circular dichroism (CD) signal is not given in units of molar ellipticity, but the CD spectra for each panel are compared on the basis of constant protein concentration
Circular dichroism spectroscopy is a powerful tool for monitoring changes in the structure of encapsulated biomolecules prepared by the sol-gel technique
Summary
The characterization of biomolecules in dilute solution is useful for determining the properties of the purified molecule, this approach fails to account for excluded volume and hydration effects that occur in concentrated, non-ideal solutions [1]. In 1992, it was first demonstrated that proper buffering of the sol–gel mixture allows one to encapsulate purified proteins without detectable denaturation [8]. Since this prominent paper, many other proteins have been encapsulated in sol-gel glasses for which protein selection has been motivated primarily by potential applications in biosensor and biocatalyst development [9,10,11,12,13,14]. Polypeptide molecules occupy individual pores as monomers, the pore walls will prevent any interactions with neighboring polypeptides that lead to aggregation This attribute of silica entrapment is demonstrated here with two disease-associated polypeptides, polyglutamine and a mutant variant of human Cu-Zn superoxide dismutase
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