Surface immobilization of engineered hemeproteins: Orientation, stability, structure, and function
Jiang, Min
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Permalink
https://hdl.handle.net/2142/22253
Description
Title
Surface immobilization of engineered hemeproteins: Orientation, stability, structure, and function
Author(s)
Jiang, Min
Issue Date
1995
Doctoral Committee Chair(s)
Sligar, Stephen G.
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Language
eng
Abstract
Biologically derived macromolecules have drawn great interests for their potentials in biotechnology and material applications. Hemeproteins, including myoglobin, cytochrome b$\sb5$ and cytochrome b$\sb{562}$ that are used in this study, are excellent candidates for the fabrication of novel biomaterials due to their unique electronic and optical properties. This work shows that protein engineering technology coupled with recently developed self-assembly and Langmuir-Blodgett techniques can provide powerful tools to manipulate micro-structures of protein monolayers at solid/air and liquid/air interfaces.
Self-assembled protein monolayers were formed on SiO$\sb2$ substrates through covalent linkage between a sulfhydryl group of a cysteine residue on protein surface and a thiol specific functional group on substrates derivatized with silane coupling agents. Hydration levels of the substrate surface showed great influence on the formation of monolayer of silanes. To increase binding ability and specificity, different linker and crosslinker molecules and various reaction conditions for protein immobilization were investigated. Protein surface activities, including both physisorption and chemisorption, were found to be largely dependent on protein surface structure.
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.
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