Joe O'Neil
Department of Chemistry
Room 520 H Parker Building
University of Manitoba
144 Dysart Rd.
Winnipeg, Manitoba, Canada, R3T 2N2
Tel (204) 256-0361
Fax (204) 474-7608
E-mail: joe.oneil@umanitoba.ca
TEACHING:
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. 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.
2. 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.
3. 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.
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.
NMR Laboratory
Mathematica Notebooks
http://home.cc.umanitoba.ca/~joneil/
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