Author: Haozhen Hu
Hu, Haozhen, 2020 Develop new technologies for protein structure manipulation and monitoring to better understand amyloidosis diseases, Flinders University, College of Science and Engineering
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This thesis aims to manipulate and monitor protein unfolding/refolding for contributing to the potential development of amyloidosis disease treatment and detection. Due to the low molecular weight and comparatively simple structure, protein beta-lactoglobulin is chosen in this study. Manipulation of the protein folding state is accomplished by using the vortex fluidic device (VFD) and high concentration of chaotropic agent guanidine hydrochloride (GuHCl), comparing to the traditional ways of refolding protein, VFD has the advantages of low cost and easy to control (from rotation speed or tilt angle). For the protein structure detection and monitoring, the conventional way by using circular dichroism (CD) and the novel way by using aggregation-induced emission (AIE) fluorescence are applied. Among them, fluorescence is believed to be relatively cheaper and giving information more directly so it is more convenient for this usage, moreover, the dye of AIE fluorescence shows great reactivity and sensitivity.
The project starts with establishing analyzing protocol, the lower negative ellipticity at 216nm of CD measurement and higher fluorescence intensity at 470nm indicate larger protein denaturation extent. The experimental parameter settings include chemical concentrations, reaction time, and VFD settings. Since protein concentration and VFD settings have been determined in the previous studies, the denaturant concentration 4M and AIE labelling time 2.5 hours are investigated and used in this project.
VFD was performed for both refolding and unfolding experiments, the results show that VFD can effectively refold the unfolded protein, as well as boost the reaction of denaturant and protein to unfolded protein. A combination experiment is designed to demonstrate the conditions for those two processes, which further proves that when denaturant is present in the solution, the two mechanisms would both existed, however, denaturation would still be dominant. To have a reference for the analysis result of the CD data, DichroWeb is introduced in this project, which is an online analysis tool for protein circular dichroism spectra, a convincing result has been obtained.
Finally, future works are proposed for optimizing the experimental result and avoiding the error caused by uncertainty factor, including the fluid behavior modification by changing VFD rotational speed and the fluorescence blue shift due to disordered protein aggregation.
Keywords: Protein folding, VFD, fluorescence, CD
Subject: Engineering thesis
Thesis type: Masters
Completed: 2020
School: College of Science and Engineering
Supervisor: Youhong Tang