Correlation of Structure and Function of Zinc metalloproteins via Solid-state NMR Methods
EMSL Project ID
7800
Abstract
In the past, we have utilized surrogate probes to address structure/function relationships for zinc. At a qualitative level the surrogate strategy works well. However, the critical nature of zinc in biology requires a detailed understanding of the chemistry, structure, and bonding of these zinc complexes and how, in turn, these manifestations alter the chemistry at the metal binding site. Hence, a qualitative understanding is not sufficient, and a direct probe of zinc is required. X-ray crystallography has been the dominant method because of the unfavorable spectroscopic properties associated with both Mg2+ and Zn2+. To be able to address this specific point, we have employed low temperature (10K) solid-state NMR utilizing cross polarization (CP) from protons to magnesium or zinc. Magnesium and zinc have similar NMR properties: both are quadrupolar with nuclear spin of 5/2 and comparable sensitivity for an equal number of nuclei. The magnetic resonance parameters for both metals are sensitive to the nature of bound ligands. This sensitivity arises from the fact that the dominant interaction in the NMR spectroscopy of these nuclides is the electric field gradient at the nucleus in question. The electric field gradient originates when there is a charge separation and the symmetry around the metal site is not Td or Oh. The origin of the charge separation is due to the nature of the bonding of these ions to their ligands (significant amount of ionic character to the bonds). This partial charge separation is further augmented by ligands that change charge (water and hydroxide) depending on the stage in the particular reaction scheme the protein is undergoing. The principal observable in a 67Zn NMR experiment will be the quadrupole coupling constant, Cq. This coupling constant is directly proportional to the electric field gradient at the Zn2+ ion; as a direct result of these relations, Cq values should be sensitive to the field gradient changes associated with water or hydroxide ligation to Zn2+. Analysis of the lineshape leads to the determination of Cq. Hence, a quadrupolar nuclide should be exquisitely sensitive to changes in structure and bonding at these sites. We have focused on the delineation of structure/function relationships in zinc metalloproteins and specifically on ZnOH2 and ZnSR functional groups. Many zinc enzymes utilize zinc bound water as a critical component of a catalytic reaction. The Zn2+ activates water through ionization, polarization, or simple displacement depending upon the mechanism. The mechanism is determined primarily by the influence of directly bound Zn-ligands, as well as hydrogen bonding with a secondary coordination sphere of side chains and/or bound waters within the protein. With these goals in mind we are examining carbonic anhydrase and some mutant CA?s as examples of water activation. In the other situation zinc promotes the nucleophilicity of the sulfur ligand, as exemplified by the E. coli. DNA repair protein Ada, (Adaptive response to alkylation). This proposal is to develop the needed spectroscopic tools for these experiments and then applies these methods to resolve critical questions relating to the role of the metals in their respective chemistries.
Project Details
Project type
Capability Research
Start Date
2004-12-29
End Date
2005-10-05
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members