Studies of Protein Conformation at Solid/Liquid and Air/Liquid Interfaces by Surface Plasmon Resonance and Langmuir-Blodgett Trough
| Autor: | Ming-Chia Wu, 吳旻珈 |
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| Rok vydání: | 2004 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 92 Behaviors of proteins immobilized on a gold surface were investigated using a home-made biosensor utilizing surface plasmon resonance(SPR). In order to examine the possibility of the use of SPR for high sensitive, real-time and label-free monitoring of the protein conformation of unfolding and refolding process, the protein immobilized on a self-assembled monolayer (SAM) layer was analyzed. Specific structure was assigned to the changing surface plasmon resonance(SPR) signals of the immobilized proteins using Langmuir-Blodgett Trough(L-B Trough), Circular Dichroism(CD) and Fluorescence spectroscopies, which are established techniques for the conformational analysis of proteins. For the oxidized proteins, from L-B trough, CD and fluorescence infer that the hydrophobic region was not exposed and retain most of the ordered secondary and tertiary structure. The denaturants(i.e. urea and guanidine hydrochloride) were bound to the protein at specifically hydrophobic sites, therefore SPR angle shift was increasing with concentration of denaturants. The reducing agent affect the conformational changes of proteins in SPR signal, make a positive SPR angle shift. From L-B truogh experiments reveal a compact conformation and hydrophobic region exposed unobviously, therefore, protein film became compactly and increase the refractive index of protein layers. For the reduced proteins, the denaturants not only destroy the ordered secondary and tertiary structure but also stabilize the unfolded conformation, that would increase the thickness of the protein layers and decrease the refractive index of the protein layers, and therefore SPR angle shift was decreasing with the concentration of denaturants. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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