In bacteria, mechanosensitive ion channels are essential for the cellular response to hypoosomitic shock, relieving the build up of membrane pressure. This thesis focuses on the Mechanosensitive Channel of Small Conductance (MscS) for which three conflicting gating models exist. These models were generated from structural studies of the closed and open conformations of MscS using three different experimental techniques. Pulsed Electron Double Resonance (PELDOR) spectroscopy was applied to MscS in the detergent solubilised state and the membrane-like bilayer. The distances between selectively introduced unpaired electrons in MscS were measured. PELDOR data in the detergent solubilised state were only consistent with the crystal structure depicting an open conformation of MscS, indicating that the transmembrane helices were unperturbed during crystallisation in detergent. MscS was reconstituted into membrane bilayer mimics, bicelles and nanodiscs, and PELDOR data in these environments suggested that both closed and open conformations determined by X-ray crystallography are stabilised in the membrane bilayer. The second part of this thesis involved stabilisation of an enzyme complex of the ubiquitin (Ub) pathway for structural analysis. This pathway is a eukaryotic signalling system involving post-translational modification of target protein amino groups with Ub. The variety of modification provided by Ub and its interplay with other Small Ubiquitin-like Modifier (SUMO) signalling proteins controls an array of cellular responses. The pathway functions to activate the Ub C-terminus, forming highly reactive thioester conjugates between Ub and the active site cysteine of a series of enzymes: E1, E2 and in some cases E3. This thesis explores the use of an isopeptide linkage to stabilise an E2~Ub conjugate in complex with an E3 enzyme. Sample conditions were optimised for the future use of PELDOR spectroscopy to structurally analyse the E2~Ub alone and in complex with E3.

Chan Ling Nga. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 143-153). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.vi / Table of Contents --- p.vii / List of Figures --- p.xi / List of Tables --- p.xiv / Abbreviations --- p.xv / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- The Human Endometrium --- p.1 / Chapter 1.1.1 --- The Structure of the Endometrium --- p.1 / Chapter 1.1.2 --- Cyclic Changes in the Endometrium --- p.1 / Chapter 1.1.3 --- Physiological Roles of the Endometrium --- p.5 / Chapter 1.1.4 --- Roles of Luminal Epithelium in Implantation --- p.5 / Chapter 1.1.5 --- Exocrine Functions of the Endometrial Epithelium --- p.6 / Chapter 1.2 --- Review of Epithelial Ion Channels --- p.8 / Chapter 1.2.1 --- Epithelial Na+ Channels (ENaC) in Absorbing Epithelia --- p.9 / Chapter 1.2.2 --- Epithelial C1- Channels in Secretory Epithelia --- p.13 / Chapter 1.2.3 --- Na+ and C1- Channels in Endometrial Epithelia --- p.15 / Chapter 1.3 --- Review of the Intracellular Signal Transduction Pathways --- p.15 / Chapter 1.3.1 --- The cAMP-Mediated Signal Transduction Pathway --- p.17 / Chapter 1.3.2 --- The cAMP-Mediated Chloride Channels in Epithelial Cells --- p.17 / Chapter 1.3.3 --- Ca2+-Dependent Signal Transduction Pathway --- p.21 / Chapter 1.4 --- Physiological Roles of some Neurohormonal Agents in Uterine Functions: Selected Examples --- p.23 / Chapter 1.4.1 --- Roles of Adrenaline on the Endometrial Ion Transport --- p.23 / Chapter 1.4.2 --- Prostaglandin (PG) E2 and PGF2α --- p.24 / Chapter 1.4.3 --- Biological Effect of Extracellular Nucleotides --- p.26 / Chapter 1.5 --- Objective of this Study --- p.28 / Chapter 2 --- Materials and Methods --- p.31 / Chapter 2.1 --- Materials --- p.31 / Chapter 2.1.1 --- Culture Media and Enzymes --- p.31 / Chapter 2.1.2 --- Drugs --- p.31 / Chapter 2.1.3 --- Chemicals --- p.32 / Chapter 2.1.4 --- Experimental Tissues and Animals --- p.32 / Chapter 2.2 --- Preparations --- p.32 / Chapter 2.2.1 --- Previous Support for Cell Growth --- p.32 / Chapter 2.2.2 --- Growth Medium --- p.33 / Chapter 2.2.3 --- Culture of Mouse Endometrial Epithelial Cells --- p.35 / Chapter 2.2.4 --- Solutions for the Short-Circuit Current Measurements --- p.36 / Chapter 2.2.5 --- Solutions for the Patch-Clamp Experiments --- p.38 / Chapter 2.2.6 --- Running Buffers for RNA and DNA Gel Electrophoresis --- p.39 / Chapter 2.2.7 --- UTP-free UDP --- p.40 / Chapter 2.2.8 --- Electrodes for the Short-Circuit Current Measurement --- p.40 / Chapter 2.3 --- Protocols --- p.41 / Chapter 2.3.1 --- Characterization of Neurohormonal Agents-induced Ion Channels --- p.41 / Chapter 2.3.2 --- Possible Interaction between CFTR and ENaC --- p.41 / Chapter 2.3.3 --- Characterization of Pyrimidinoceptors-mediated Conductances --- p.42 / Chapter 2.4 --- Methods of Measurements --- p.42 / Chapter 2.4.1 --- The Patch-Clamp Technique --- p.42 / Chapter 2.4.1.1 --- The Patch-Clamp Setup --- p.43 / Chapter 2.4.1.2 --- Shielding and Grounding --- p.45 / Chapter 2.4.1.3 --- Pipette Fabrication --- p.45 / Chapter 2.4.1.4 --- Pipette Holder and Electrodes --- p.48 / Chapter 2.4.1.5 --- Experimental Procedures --- p.49 / Chapter 2.4.1.6 --- Signal Recording and Data Acquisition --- p.54 / Chapter 2.4.1.7 --- Data Analysis --- p.54 / Chapter 2.4.2 --- The Short-Circuit Current Technique --- p.55 / Chapter 2.4.2.1 --- The Short-Circuit Current Setup --- p.56 / Chapter 2.4.2.2 --- Experimental Procedures --- p.56 / Chapter 2.4.2.3 --- Data Analysis --- p.61 / Chapter 2.4.3 --- Reverse Transciption - Polymerase Chain Reaction (RT-PCR) --- p.61 / Chapter 2.4.3.1 --- RNA Isolation --- p.61 / Chapter 2.4.3.2 --- RNA Gel Electrophoresis --- p.62 / Chapter 2.4.3.3 --- Reverse Transcription (RT) --- p.63 / Chapter 2.4.3.4 --- Polymerase Chain Reaction (PCR) --- p.64 / Chapter 2.4.3.5 --- DNA Gel Electrophoresis --- p.66 / Chapter 2.4.4 --- Statistical Analysis --- p.66 / Chapter 3 --- Results --- p.67 / Chapter 3.1 --- Activation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in Response to Hormonal Stimuli --- p.67 / Chapter 3.2 --- Inhibition of Na+ Absorption by CFTR --- p.89 / Chapter 3.3 --- Pyrimidinoceptors-activated Ca2+-dependent C1- Conductance --- p.111 / Chapter 4 --- General Discussions --- p.132 / Appendix --- p.140 / Chapter A --- RNA Isolation --- p.140 / Chapter B --- Reverse Transcription --- p.141 / Chapter C --- Polymerase Chain Reaction --- p.142 / References --- p.143