Department of Chemistry
Room 390 Parker Building
University of Manitoba
144 Dysart Rd.
Winnipeg, Manitoba, Canada, R3T 2N2
Tel (204) 474-6697
Fax (204) 474-7608
The main focus of my research is to understand the molecular structural basis of the biological activities of proteins. We use Nuclear Magnetic Resonance (NMR) spectroscopy, Circular Dichroism (CD) spectropolarimetry, molecular biology, and hydrogen exchange chemistry to measure the structures and dynamics of proteins, and to relate the information obtained to the actions of the proteins. The proteins under investigation are the membrane proteins, alamethicin and glycerol facilitator, and the intrinsically disordered protein, HIV-1 Transactivator of transcription.
1. Glycerol facilitator: Membrane proteins comprise about 25% of all prokaryotic and eukaryotic proteins yet studies of their structure and folding lag far behind those of water-soluble globular proteins. Glycerol facilitator is an intrinsic membrane protein found in E. coli and belongs to a large class of proteins that function to enhance the transport of polar solutes across non-polar membranes. We have developed an expression and purification system that has permitted us to study its secondary, tertiary, and quaternary structure and stability in several different detergents. On the right, is a CD spectrum of the protein tetramer showing aromatic side-chain bands that are characteristic of the folded protein. We are exploring these preparations for the application of NMR spectroscopy.
2. Alamethicin: This fungal antibiotic peptide inserts into membranes and forms voltage-gated ion channels. We have investigated its structure and dynamics dissolved in methanol and detergent micelles and have completed a high-resolution structure determination of alamethicin in methanol. This work was possible because we developed methods to biosynthetically incorporate 15N and 13C into alamethicin and this was the first such labeling of a fungal protein. To gain insight into its function as a pore molecule we have synthesized dimers of the peptide that have been studied by NMR and CD. We have also collaborated with Professor Burkhard Bechinger (Univ. Louis Pasteur, Strasbourg) to determine, using solid-state NMR spectroscopy, the structure of alamethcicin in a lipid bilayer.
3. Tat: The Transactivator of transcription (Tat) is a small RNA-binding protein that plays a central role in the regulation of HIV-1 replication and in approaches to treating latently-infected cells. To increase the dispersion of NMR signals and to permit dynamics analysis by multinuclear NMR spectroscopy we have prepared uniformly 15N and 15N /13C-labelled Tat1-72 protein. Our high-resolution NMR analyses of the dynamics of Tat show that it is an intrinsically disordered protein.
4. Model Amides: We synthesized a series of hydrophobic model amides and studied their mechanisms of hydrogen-deuterium exchange by 1H NMR spectroscopy. This work showed for the first time that detergent micelles can slow the exchange process by restricting access of catalytic solvent to the interior of the micelle. This result has important implications for the study of hydrogen exchange of proteins in detergent.
List of Publications
Maintained by J. O'Neil
Maintained by J. O'Neil