Correlation of Structure and Function of Zinc Metalloproteins Via Solid-state NMR Methods
EMSL Project ID
10597a
Abstract
As early as 1869 it was known that zinc is an essential element for eukaryotes, it is the second most abundant trace element in humans after iron. About half of all proteins contain metal cations, which play predominantly a catalytic or structural role. 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. A qualitative understanding is not sufficient; a direct probe of zinc is required. X-ray crystallography has been the dominant method because of the unfavorable spectroscopic properties associated with 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 the zinc. The magnetic resonance parameters for zinc are sensitive to the nature of bound ligands. This sensitivity arises from the fact that the dominant interaction in the NMR spectroscopy of this nuclide is the electric field gradient at the nucleus in question (zinc is quadrupolar; spin 5/2). 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. Analysis of the NMR 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 alkaline phosphatase 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).
It has become apparent from our past work that the secondary ligation sphere and solvation effects are critical to modeling an active site. Our current model for alkaline phosphatase contains 329 atoms and utilizes close to 1800 basis functions. This one calculation has used more than 40000 CPU hours on PNNL?s SGI Altix and is just approaching convergence on the geometry. Hence our need to scale up our calculations in an effort to accurately describe the necessary components of the model.
As a by-product of this research we will gain an understanding of the structure/function relationships for metalloproteins in general. This can have applicability for other metals such as mercury, magnesium, lead, and/or iron. This can also have implications for bioremediation or heavy-metal trafficking within microbial communities. The first step in understanding how to fine-tuning a system or how metals poison a system (i.e. co-carcinogenesis) is comprehension of the native systems, which is the work we are undertaking.
Project Details
Project type
Capability Research
Start Date
2005-04-11
End Date
2006-04-17
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members