Structure-Function Correlation of the M2 Proton Channel Characterized by Solid-State Nuclear Magnetic Resonance Spectroscopy

• Other Authors: Hu, Jun (authoraut)
• Format: Others
• Language: English; English
• Published: Florida State University
• Subjects: Chemistry
• Online Access: http://purl.flvc.org/fsu/fd/FSU_migr_etd-3695

Although still in a developing age, solid-state nuclear magnetic resonance (NMR) spectroscopy has been proved to be a powerful tool to study the structure and dynamics of membrane proteins. Here this technique is applied to investigate the channel conductance mechanism and inhibition of the M2 proton channel from the influenza A virus. A four-histidine cluster in the pore of the M2 proton channel has been characterized by cross-polarization magic angle spinning (CP/MAS) NMR experiments over a pH range from 5 to 8.6. Studies were carried out in fully hydrated lipid bilayers with d1 and e2 15N labeled imidazole side chains. The first two protons bind to this histidine cluster with high affinity into imidazole-imidazolium dimeric forms with nearly identical pKas of 8.2 suggesting the possibility of cooperative H+ binding. The resulting 'histidine lock' formed by a pair of imidazole-imidazolium dimers occludes the pore thereby closing the channel. The acid activation of the channel, which has long been associated with a histidine titration, is now associated with the third charge in this cluster that disrupts the 'histidine lock.' The H+ selectivity for the channel is explained by either a Grotthüs, water-wire or a histidine shuttle mechanism. Both side chain and backbone NMR data indicate that amantadine, an anti-viral drug, appears to hinder the M2 tetramer dynamics. The structure of the M2 transmembrane helices is determined by a static 2D solid-state NMR technique, polarization inversion of spin exchange at magic angle (PISEMA). Waves, wheels and helical spectral patterns are observed in PISEMA data. Two PISA wheels in the PISEMA spectra demonstrate a helical bend in the transmembrane domain of the M2 protein in the presence of amantadine. === 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, 2005. === January 24, 2005. === The M2 Protein, Solid-State NMR, Proton Channel, Amantadine, Influenza, PISEMA, CPMAS === Includes bibliographical references. === Timothy A. Cross, Professor Directing Dissertation; Peter G. Fajer, Outside Committee Member; Michael S. Chapman, Committee Member; Naresh Dalal, Committee Member; Timothy M. Logan, Committee Member.