Probing Structures of Membrane Proteins and Their Inhibitors

• Main Author: Kim, Chang Gyeom
• Published: University of Oxford 2005
• Subjects: 572
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.491627

Using solid-state NMR and computational approaches, this study examines the binding-site conformations of inhibitors of membrane proteins such as the gastric H+/K+-ATPase, an integral membrane protein that represents the major target in the treatment of gastric ulcer disease, oesophageal acid reflux, and duodenal ulcer disease, an acetylcholinesterase (AChE) that has an important role in signal transduction in a nerve cell, and a Vpu which is a HIV-l viral protein. Solid-state NMR techniques are applied to the study of these compounds, which are derivatives of the substituted imidazopyridine SCH28080, to refme the ligand binding site and to develop more accurate protein models. 2HYF and 19F_13C REDOR NMR experiments are employed to measure the distance between site-specific substitutions in the inhibitors. Using symmetry-based R sequence, a new DQF pulse sequence (RI6:> which can be applied to a high spinning MAS experiment is developed and applied to the complex of E/edrophomJ e/eclrit:1lJ acetylcholinesterase (EeAChE) and its inhibitor, to probe the chemical environments of the bound inhibitor. Protein 3D structure models are created using MODELLER for two conformers of the gastric H+/K+-ATPase, in order to confirm the experimental data, to study its threedimensional structure, and to provide details about inhibitor bound to the protein. The Ca2+-ATPase is chosen as a template for the gastric H+/K+-ATPase. For probing the protein-ligand interaction, docking simulations are performed using AutoDock3. The simulations revealed different results according to the catalytic (E, or EJ state of the H+/K+-ATPase. In the E, state, inhibitors bound lumenally at the TMS-Thf6 interhelical loop and proximal to TM6. In the E2 conformation, the inhibitor bound consistently in the interhelical space which is proposed to be the site ofion translocation. For the Vpu, the binding site for two ion channel blockers, arniloride (AM) and cyclohexamethylenearniloride (HIvfA), are examined by using a protein-ligand docking approach. The three different protonation states of both inhibitors are tested for the docking simulations and the simulations show that the inhibitor binds to the Ser23 through the hydrogen bond interaction.