Zinc and Magnesium NMR of Systems of Biological Interest
Paul Ellis, Principal Investigator
Funding Agency: National Institutes of Health, National Institute of Biomedical Imaging and Bioengineering
The Zinc and Magnesium NMR for Systems of Biological Interest research team at Pacific Northwest National Laboratory (PNNL) is continuing to establish a correlation between 67Zn and 25Mg magnetic resonance parameters and the structure-function relationships for known Zn- and Mg-metalloproteins. In addition, we are working to extrapolate those relationships to proteins where the role of the metal is less defined. To this end, we are continuing to develop the needed spectroscopic tools for these experiments and to apply these tools to resolve critical questions relating to the role of the metals in their respective chemistries. The scientific aims being addressed as part of our research include:
- compare the 67Zn NMR parameters of a mutant human carbonic anhydrase with bound HCO3- and a synthetic analogue
- characterize the 67Zn NMR spectroscopy of the synthetic analogue of water bound to the zinc in human carbonic anhydrase
- establish a baseline understanding of the role of the 67Zn quadrupole coupling constants in a variety of Zn(Cys)4-containing proteins and compare those results to measurements of E. coli Adaptive response to alkylation (Ada) protein with and without binding to its consensus DNA sequence
- characterize the 67Zn NMR spectroscopy of alkaline phosphatase and several mutants to assign the resonances for the two zinc sites and determine the nature of the ligands bound to both zinc sites as a function of pH and reaction conditions
- characterize the 25Mg NMR spectroscopy of alkaline phosphatase and several mutants as a function of pH and reaction conditions to better define the role of Mg2+ in this protein.
To understand the role of the Mg2+ and Zn2+ in native proteins or to engineer a Zn2+ catalyst with predictable structure and function, one must first be able to characterize the structure and bonding at the metal site. Historically, the only reliable method for such a characterization of a zinc site has been through X-ray crystallography. However, the X-ray crystallography of magnesium-containing proteins has a complication: it is difficult to distinguish Mg2+ and O2- in modest resolution protein structures because of the isoelectronic nature of the cation and anion. Further, positional disorder can lead to confusion between disordered water and the presence of Mg2+. The NMR spectroscopy of Mg2+ and Zn2+ in their native proteins should provide information complementary to that obtained from X-ray structures.
