Initial structural characterization of the integrated membrane M2 proton channel from influenza A virus using solid state and solution state NMR

Structure biology of membrane proteins is of great interest because they are responsible for most of the dynamic processes across biological membranes including ion/molecular transport, communication and energy transduction. Solid state NMR of membrane proteins reconstituted into uniformly aligned lipid bilayers has been developed and applied to obtain atomic resolution structural information for membrane proteins and recently several complete 3D structures of membrane associated polypeptides have been solved using this method. However, solid state NMR studies of full-length membrane proteins are different from studies of membrane associated polypeptides in isotope labeling, sample preparation, sample stability and experimental sensitivity. A transition from solid state NMR structural studies of membrane associated polypeptides to those of full-length membrane proteins has been made in this dissertation resulting in new protocols, new methods for membrane protein synthesis, isotope labeling, sample preparation, sample characterization and solid state NMR experiments.; M2 protein from influenza A virus has been over-expressed in E. coli, purified and reconstituted into lipid bilayers, where it forms a stable tetrameric preparation. 15N uniform labeling and 15N amino acid specific labeling of M2 protein have been achieved for 15N-1H dipolar/15N chemical shift correlation PISEMA (Polarization Inversion Spin Exchange at Magic Angle) experiments. The reconstituted M2 protein in lipid bilayers was used for preparing uniformly aligned solid-state NMR samples. PISEMA spectra of these samples support a transmembrane helix in M2 protein having a tilt angle of approximately 25° to the bilayer normal and further suggest the possibility for a complete backbone structure determination of the transmembrane helical segment. In addition, hydrogen-deuterium exchange experiments, solid state NMR PISEMA experiments and PISA (Polar Index Slant Angle) wheel analysis suggest an aqueous pore is present in the proton channel and an 80° tilted amphipathic helical segment half-buried in the lipid-bilayer/aqueous environment interfacial region. The chemically synthesized transmembrane peptide of the M2 proton channel (M2-TMP) has been reconstituted in detergent micelles and solution NMR study of this preparation indicates a pore-formation from a tetrameric structure.