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- All Subjects: Biochemistry
- Creators: Van Horn, Wade
- Resource Type: Text
The x-ray crystallography structure of the β clamp suggests that there are oppositely charged amino acid pairs present at the interface of the dimer. They can form strong electrostatic interactions between them. However, for Proliferation Cell Nuclear Antigen (PCNA), there are no such charged amino acids present at its interface. High sodium chloride (NaCl) concentrations were used to disrupt the electrostatic interactions at the interface. The role of charged pairs in the clamp interface was characterized by measuring the apparent diffusion times (\tau_{app}) with fluorescence correlation spectroscopy (FCS). However, the dissociation of the Proliferation Cell Nuclear Antigen (PCNA) trimer does not depend on sodium chloride (NaCl) concentration.
In the next part of my thesis, potassium glutamate (KGlu) and glycine betaine (GB) were used to investigate their effect on the stability of both clamp proteins. FCS experiments with labeled β clamp and Proliferation Cell Nuclear Antigen (PCNA) were performed containing different concentrations of potassium glutamate and glycine betaine in the solution, showed that the apparent diffusion time\ {(\tau}_{app}) increases with potassium glutamate and glycine betaine concentrations, which indicate clamps are forming higher-order oligomers. Solute molecules get excluded from the protein surface when the binding affinity of the protein surface for water molecules is more than solutes (potassium glutamate, and glycine betaine), which has a net stabilizing effect on the protein structure.
To gain more information about the function of the transmembrane region of hTRPM8, it was expressed in Escherichia coli (E. coli) and purified in detergent membrane mimics for experimentation. The construct contains the S4-S5 linker, pore domain (S5 and S6 transmembrane helices), pore helix, and TRP box. hTRPM8-PD+ was purified in the detergents n-Dodecyl-B-D-Maltoside (DDM), 16:0 Lyso PG, 1-Palmitoyl-2-hydroxy-sn-glycero-3-phosphoglycerol (LPPG), and 14:0 Lyso PG, 1-Myristoyl-2-hydroxy-sn-glycero-3-phosphoglycerol (LMPG) to determine which detergent resulted in a hTRPM8-PD+ sample of the most stability, purity, and highest concentrations. Following bacterial expression and protein purification, hTRPM8-PD+ was studied and characterized with circular dichroism (CD) spectroscopy to learn more about the secondary structures and thermodynamic properties of the construct. Further studies can be done with more circular dichroism (CD) spectroscopy, planar lipid bilayer (BLM) electrophysiology, and nuclear magnetic resonance spectroscopy (NMR) to gain more understanding of how the pore domain plus contributes to the activity of the whole protein construct.
In intracranial aneurysms, multiple factors and biochemical pathways are believed to be involved in the event of a rupture. The epidermal growth factor receptor (EGFR) activation pathway is of particular interest as a way to understand and target the mechanism of rupture due to its established role in cellular proliferation and inflammation. Furthermore, unfolded protein responses in vascular cells’ endoplasmic reticulum (ER), known as ER stress, have emerged as a potential downstream mechanism by which inflammatory EGFR activation may lead to aneurysm rupture. The purpose of this project was to investigate the role of EGFR inhibition on the aneurysm rupture rate in a preclinical model, investigate the role of ER stress induction on the aneurysm rupture rate, and confirm which cellular phenomenon lies upstream in this mechanistic cascade. Based on analyses of aneurysm rupture rate and gene expression in the Circle of Willis, ER stress and inflammatory unfolded protein responses were found to be downstream of initial EGFR activation, which may be an effective therapeutic target for preventing aneurysm rupture in a clinical setting.