"Biophysical Studies of Human Aquaporin 1 Structural Insights by Solid-State NMR and Mechanism of Inhibition by Mercury"
Human Aquaporin 1 (hAQP1) is a membrane protein that transfers water through the membrane with a rate faster than that of the simple diffusion. This protein plays an important role in different tissues such as the kidney, the eyes and the skin in human body. Overexpression of this protein can be associated with different diseases such as cancer, glaucoma, and brain swelling. Thus, inhibition of hAQP1 is a promising approach to decrease the symptoms of such diseases and, ultimately, to cure them. Therefore, knowing the function and structure of the protein in its native environment is important in order to understand the inhibitory effects of different drugs. In this respect, solid-state NMR (ssNMR) is a promising technique as it can probe structure and dynamics of membrane proteins in lipids. In order to study hAQP1 with ssNMR, the most critical step is to overexpress isotopically labeled functional protein in its proper fold with good stability. In this project, the production of the doubly (13C/15N) isotopically labeled hAQP1 protein in yeast Pichia pastoris was established. The production of homogeneous labeled protein yielded an excellent resolution of the ssNMR spectra, which allowed running suites of multidimensional experiments yielding assignments for the majority of hAQP1 resonances. The assignments revealed a wealth of site-specific information, including the secondary structure, chemical environment, hydrogen bonding and water accessibility of the amino acid residues (via H/D exchange). The inhibition studies of hAQP1 by a mercurial compound were also conducted using different biophysical techniques such as ssNMR, Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), small angle X-ray scattering (SAXS) and stopped-flow kinetic experiments. The novel mechanism of inhibition of hAQP1 by mercury chloride was shown to affect its conformation and to cause the tetramer and lattice disruption. Finally, novel promising organic compounds were tested and shown to be effective as inhibitors in vitro, using proteoliposome systems.
Dr. Hermann Eberl, Chair (Department of Mathematics and Statistics)
Dr. Vladimir Ladizhansky, Advisory Committee (Department of Physics)
Dr. Leonid Brown, Advisory Committee (Department of Physics)
Dr. George Harauz, Examination Committee (Department of Molecular & Cellular Biology)
Dr. Isabelle Marcotte, External Examiner (Université du Québec à Montréal, Montréal, QC)