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40835-AC3
Investigation of the Factors Affecting Reactivity of Thiolate-Rich Zn(II) Sites in Proteins

Gregg R. Dieckmann, University of Texas (Dallas)

Cysteine-rich Zn(II)-binding sites in proteins serve two distinct functions: to template or stabilize specific protein folds, and to facilitate chemical reactions such as alkyl transfers. We are interested how the protein environment controls metal site properties, specifically, how naturally occurring tetrahedral Zn(II) sites are affected by the surrounding protein. Our first step has been to identify a reasonable model system to study metal-protein interactions. For this purpose, we have studied the Co(II)- and Zn(II)-binding of a series of derivatives of L36, a small 37-residue zinc ribbon protein containing a Cys₃His metal coordination site. L36 has been structurally characterized previously (NMR), and is easily synthesized using standard solid phase peptide synthetic methodologies. UV-Visible spectroscopy was used to monitor metal binding by peptides at pH 6.0. For all derivatives, the following trends were observed: (1) Zn(II) binds tighter than Co(II); (2) mutation of the metal-binding ligand His32 to Cys decreases the affinity of L36 derivatives for both metals; (3) a Tyr24 to Trp mutation in the beta-sheet hydrophobic cluster increases K_A^Zn and K_A^Co; (4) mutation in the beta-hairpin turn, His20 to Asn generating an Asn-Gly turn, also increases K_A^Zn and K_A^Co; (5) the combination of His20 to Asn and Tyr24 to Trp mutations also increases K_A^Zn and K_A^Co, but the increments vs. the native L36 sequence are less than those of the single mutations. Furthermore, circular dichroism, size exclusion chromatography, and 1-D and 2-D ¹H NMR experiments demonstrated that the mutations do not change the overall fold or association state of the proteins. Recent work has also focused on a new set of mutations which involve keeping the Cys₃His binding site intact, but changing the sequence location of the His (similar to “rotating” the binding site in the context of the protein). Interestingly, these derivatives show modulations in their electronic spectroscopic characteristics when binding Co(II); we are in the process of fully characterizing their metal-binding affinities and 3-dimensional structural properties. L36, displaying Co(II)- and Zn(II)-binding sensitivity to various sequence mutations without undergoing a change in protein structure, can therefore serve as a useful model system for structure/reactivity studies.

We are conducting detailed methylation studies for the L36 derivatives, the goal of which is to correlate differences in reactivity with methyl transfer reagents to sequence mutations we introduce into the protein. Methylation experiments utilize anaerobic conditions and one of three methylation agents (selected because all have been used previously in the study of methylation of Cys residues in proteins and, specifically, metal-bound thiolates): trimethyloxonium tetrafluoroborate (TMO), dimethyl sulfate (DMS) or CH₃I. We are monitoring the methylation reactions using several techniques:

(a) changes in UV/Visible spectra upon addition of methylation agent to Co(II)-bound derivatives. For Cys₄ derivatives, we see a rapid release of Co(II) (reflected in a loss of the Co(II) d-d transitions), whereas for the Cys₃His derivatives the absorbance does not change significantly. We are currently using pre-synthesized methyl-Cys derivatives of L36 to understand if the Co(II) loss is due to methylation of a Cys in the metal-binding site, or if the anion generated from the methylation reaction extracts the Co(II) from the protein. Our results indicate that methyl-Cys L36 derivatives are capable of binding Zn(II) (albeit at higher required Zn(II) concentrations, implying a weaker binding). Binding to Co(II) was not observed for these derivatives up to a [Co(II)] of 200 millimolar. These results suggest that metal binding is weakened, as predicted, for incorporation of a methyl-Cys in the metal-binding site, indicating that methylation of a Cys ligand could be sufficient to cause metal loss.

(b) changes in the mass spec. of L36 fragments generated after methylation treatment using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) labeling of unmethylated Cys, followed by alkaline cleavage. For this, we have observed the expected cleavage of the peptides treated with CDAP, but have been unable to obtain clean and interpretable mass spec results.

(c) changes in Raman and NMR spectra.

We are also modifying the electrostatic screening at the metal site by the removal of H-bonding interactions to the Cys ligands. One of the backbone amide H-bonds is being removed using native chemical ligation and an alpha-hydroxy acid derivative of one of the amino acids to synthesize a L36 derivative with a backbone ester linkage replacing a backbone amide.

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