Design and Quantitation of Membrane Binding Lipid Anchors : Exploring Prion-Prion Interactions on Membrane Surfaces
The prion protein (PrP) is an endogenous, metal binding protein present in the neuronal cells of the central nervous system. Prion is associated with a class of neurodegenerative diseases known as transmissible spongiform encephalopathies. The C-terminal region of the prion protein is anchored to the cell surface by means of a glycophosphatidylinositol (GPI) anchor[superscript]19. Studies indicate that PrP self-recognition may be an important factor in both the normal function and misfunction of PrP. Elucidating the molecular basis for PrP-PrP interactions in the context of its membrane bound state will help in understanding the normal function of PrP, such as the signaling mechanism for endocytosis, and the factors that influence disease causing structural changes. Fluorescently labeled models of prion protein were previously developed to investigate PrP-PrP interactions and metal binding at molecular level. Peptides constituting the metal binding region were anchored to small unilamellar vesicles (liposomes) and PrP-PrP interactions were studied as a function of added metal45. Anchoring the peptides is an essential step to understand the protein interactions in the context of a cell surface. The main objective of this research is to prepare a molecule capable of anchoring the majority of a PrP sample to a liposome and develop a spin-label based assay to determine the percentage of molecules anchored to the liposome surface. Four lipophilic molecules containing a nitroxide spin-label have been synthesized and their electron paramagnetic resonance (EPR) spectra collected in the presence and absence of liposomes. The EPR spectrum of the nitroxide is very sensitive to the motion of the spin label and the proximity to other spin labeled molecules. The anchor with a linear chain of sixteen carbon atoms showed the most dramatic changes in the EPR spectrum and is likely the best anchor. We are planning to use a paramagnetic relaxation agent that aids in the quantitation and fluorescent compounds which aid in determining where the spin-labeled molecules localize. The spin-label methodology will allow us to conduct more quantitative experiments on PrP interactions with respect to metal binding, change in temperature, pH etc.
Gangula, Manasa. (January 2011). Design and Quantitation of Membrane Binding Lipid Anchors : Exploring Prion-Prion Interactions on Membrane Surfaces (Master's Thesis, East Carolina University). Retrieved from the Scholarship. (http://hdl.handle.net/10342/3736.)
Gangula, Manasa. Design and Quantitation of Membrane Binding Lipid Anchors : Exploring Prion-Prion Interactions on Membrane Surfaces. Master's Thesis. East Carolina University, January 2011. The Scholarship. http://hdl.handle.net/10342/3736. October 17, 2018.
Gangula, Manasa, “Design and Quantitation of Membrane Binding Lipid Anchors : Exploring Prion-Prion Interactions on Membrane Surfaces” (Master's Thesis., East Carolina University, January 2011).
Gangula, Manasa. Design and Quantitation of Membrane Binding Lipid Anchors : Exploring Prion-Prion Interactions on Membrane Surfaces [Master's Thesis]. Greenville, NC: East Carolina University; January 2011.
East Carolina University