 |
My group is interested in the role of transition metal ions in proteins that are involved in disease, infection and injury. The human body requires a large number of transition metal ions, including Co, Cu, Fe, Mn, Mo, Zn and Ni. These are involved in processes including energy transduction, metabolism, cell recognition, oxygen transport, protein processing, protein targeting, gene regulation and DNA/RNA processing. All of these metal ions bar Zn often exist in oxidation states containing unpaired electrons and can be detected by a magnetic resonance technique, electron paramagnetic resonance (EPR) spectroscopy. Some of the techniques we use, and systems that we work on, are summarized here. |
| Electron paramagnetic resonance (EPR). A magnetic resonance technique that provides information on the oxidation state, geometry and ligand environment of the metal centers in enzymes, and enzyme complexes and intermediates. EPR can also be used to measure distances between spins and provide detailed structural information on site-specifically 'spin-labeled' proteins. Two important aspects of our research in EPR methodology are (i) developing methods to extract structural information on zinc enzymes from EPR of substituted Co(II), and (ii) increasing the information content of EPR spectra of Co(II) and Cu(II) by optimizing the frequencies at which the spectra are recorded. This work is in conjunction with Profs Bill Antholine and James Hyde and uses computer simulation software developed by Prof Graeme Hanson. This work is partially supported by NIBIB/NIH (EB001980). |
 |
 |
Rapid-freeze-quench (RFQ). A method of rapidly mixing and freezing solutions. Using RFQ, we can trap enzyme catalytic cycle intermediates for examination by EPR ('RFQ-EPR'). By taking EPR 'snapshots' along the reaction coordinate we investigate the catalytic mechanism. By using spin-labeled enzymes, conformational changes during the enzymatic reaction can be followed. |
| Vibrio aminopeptidase (VpAP). VpAP and its homologs are zinc-dependent aminopetidases that are secreted by bacteria including Vibrio, Clostridium, and Streptomyces. The enzyme is a virulence factor in the infectivity of these bacteria. VpAP also has very close structural homology to the peptidatic domain of the human prostate specific membrane antigen. Investigation of the mechanism of VpAP provides useful information for the design of antibacterial agents and inhibitors with therapeutic, investigative and diagnostic potential with prostate cancer. Much of the work on VpAP has been carried out with Prof Rick Holz. We have recently proposed a novel hydrolytic mechanism for VpAP based on crystallography and RFQ-EPR. The work is funded by NIAID/NIH (AI056231). |
 |
 |
Metallo-b-lactamases (MBLs). MBLs confer resistance to b-lactam antibiotics upon many pathogenic bacteria. We have used EPR and RFQ-EPR to study a number of these enzymes, providing information on metal center assembly, the structures of the active metal centers, the mechanisms of catalysis, and conformational changes during catalysis. These studies provide new information relevant to the design of antibacterials toward resistant pathogens. This work is carried out in conjunction with Profs Mike Crowder, Alejandro Vila and David Tierney. |
Other projects. We also study the proteins a-synuclein (AS), xanthine oxidase (XO), nitrile hydratase (NH) and the prostate-specific membrane antigen (PSMA; GCPII). AS is involved in neurodegenerative diseases, particularly Parkinson's disease and Lewy body dementia and we are interested in the role of metal binding in fibril formation. XO is a molybdoprotein and we are investigating the interactions of nitric oxide with XO. NH contains an unusual iron center and is an important biocatalyst due to its stereospecificity. PSMA is very highly overexpressed in prostate