Biophysical Investigation of the Cataract Associated Mutant, E107A of Human Gamma-D Crystalline And Constructing a Fluorescent System for Manipulating PIP2 Concentration in the Plasma Membrane of Xenopus Oocytes.
Date of Award
Bachelor of Science
Chapter 1: Biophysical investigation of the cataract associated mutant, E107A of human gamma-D crystallin. Several human genetic cataracts are associated with mutations in a single amino acid residue of the protein known as D crystallin. Recently, the Glu107Ala (or E107A) mutation has been included in this list, and it has been associated with a nuclear congenital cataract. We have made recombinant human D crystallin and its E107A mutant and conducted preliminary comparisons of its structure and biophysical properties with that of the wild-type protein. In this process we made use of several techniques – Circular Dichroism (CD), Iso-electric focusing (IEF) gels, Dynamic Light Scattering (DLS), and Nuclear Magnetic Resonance (NMR). Results highlight small structural changes while the overall protein structure remains folded and intact. Chapter 2: Constructing a fluorescent system for manipulating PIP2 concentration in the plasma membrane of Xenopus Oocytes. Potassium channels play a key role in electric activity of excitable cells. Dysfunction of these ion channels may result in neuronal, cardiac, and muscular disorders. Several subfamilies of potassium channels are regulated via G-protein coupled receptor signaling pathway and the signaling molecule PIP2 may affect potassium channel activity. To understand how PIP2 regulates potassium channels, we are going to construct a unique fluorescent system to manipulate the PIP2 levels in the plasma membrane of Xenopus oocytes, an ideal heterologous expression system for studying function of ion channels. We plan to heterologously express iii 3 membrane-attached FRB-CFP fusion protein and cytoplamic RFP-FKBP12-phosphoinositide 5-phosphatase fusion protein in Xenopus oocytes. Application of rapamycin will result in heterodimerization of a FKBP12 protein with FRB protein. This process will increase the concentration of phosphoinositide 5-phosphatase and lower the concentration of PIP2 in the membrane of Xenopus oocytes. Accordingly the fluorescent signals will change during this process. With site-directed mutagenesis and sub-cloning strategies, we successfully made a construct that expresses RFP-FKBP12 fusion protein in Xenopus oocytes. This construct will be used as a negative control in this system. iv
Patrosz, Julita, "Biophysical Investigation of the Cataract Associated Mutant, E107A of Human Gamma-D Crystalline And Constructing a Fluorescent System for Manipulating PIP2 Concentration in the Plasma Membrane of Xenopus Oocytes." (2010). Biological Sciences. 11.