Structural and Dynamics Characterization of Membrane Proteins Using Static Solid-State Nuclear Magnetic Resonance Spectroscopy

• Other Authors: Li, Conggang, 1974- (authoraut)
• Format: Others
• Language: English; English
• Published: Florida State University
• Subjects: Chemistry
• Online Access: http://purl.flvc.org/fsu/fd/FSU_migr_etd-1437

Solid-state nuclear magnetic resonance (NMR) provides a unique approach for structure determination and functional studies of membrane proteins in a native lipid bilayer environment. Here, polarization inversion spin exchange at magic angle (PISEMA) experiments of aligned samples were applied to study the proton channel, M2 protein transmembrane portion (M2-TMD) from influenza A virus and other intact full length membrane proteins. The challenges of membrane protein structure characterization utilizing static aligned sample, including sample preparation, sample stability induced by RF heating, PISEMA experiment set up were discussed. For M2-TMD proton channel, drug bound state, closed state and open state were characterized by the PISEMA spectra of uniformly and selectively specific labeled aligned sample. The M2-TMD helices adopt 38 ° tilt with respect to the magnetic field from organic solvent preparation, while changing to 32 ° tilt angle from aqueous preparation. Upon the amantadine binding, M2-TMD backbone is more constrained and an kink of the helices is induced in the C-terminus. At low pH 5.0, the line-width is dramatically broadened due to conformational heterogeneity, which may be reflecting the various His37 charge states at this pH. Solid-state NMR line shape of a membrane protein in lipid bilayer aligned between glass-slides is a very sensitive to the local conformational change which is maybe indispensable for the function of the protein. The interaction of the antiviral drug, amantadine with the membrane and the channel was also probed by anisotropic interaction in the DMPC lipid bilayers. The drug is found to assume a single preferred orientation and location when incorporated in these bilayers. The experimental and MD computational results demonstrate that the long axis of amantadine is on average parallel to the bilayer normal and the amine group is oriented towards the hydrophilic domain of the bilayers. The localization of amantadine was determined by paramagnetic relaxation and by a 10 ns MD simulation showing that amantadine is within the interfacial region in which the amine interacts with the lipid headgroup and glycerol backbone, while the hydrocarbon portion of amantadine interacts with the glycerol backbone and much of the fatty acyl chain as it wraps underneath the drug. The lipid headgroup orientation changes upon drug binding as characterized by the anisotropy of 31P chemical shielding and 14N quadrupolar interactions. Amantadine appears to keep the same orientation as that observed in DMPC bilayers when binding to the transmembrane domain of the influenza A M2 proton channel. Initial structural characterizations of intact membrane proteins ranging from 1 to 3 transmembrane helices in different oligomeric states by high resolution PISEMA spectra have also been recorded in this dissertation. The resolution of the specific amino acid labeled samples shows promise for large membrane protein structure determination utilizing aligned samples and shows resonance patterns known as PISA wheels. The tilt angles of the transmembrane helices are extracted from the resonance patterns in PISEMA spectra. === A Dissertation Submitted to the Department of Chemistry and Biochemistry in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy. === Spring Semester, 2007. === March 27, 2007. === Proton Channel, Protein Structure, RF Heating, PISEMA, Solid-State NMR, Membrane Protein, Influenza A Virus === Includes bibliographical references. === Timothy A. Cross, Professor Directing Dissertation; Jack Quine, Outside Committee Member; Robert Fulton, Committee Member; Timothy Logan, Committee Member; Riqiang Fu, Committee Member.