The Tyrosine Photophysics of a Primase-Derived Peptide Are Sensitive to the Peptide's Zinc-Bound State:  Proof That the Bacterial Primase Hypothetical Zinc Finger Sequence Binds Zinc

Abstract

A 35-amino acid peptide corresponding to the putative "zinc finger" sequence of primase was prepared to study its zinc binding properties. When zinc was added to the peptide, it was found that the fluorescence quantum yield of the single tyrosine increased by 46% and the average lifetime by 34%. The binding stoichiometry was one zinc per peptide. Below pH 6.0 and above pH 8.5, the zinc-peptide binding affinity was less than 1 microM and could be accurately determined. Interpolation from those binding constants suggested that the affinity at pH 7.5 was between 10 and 100 nM. The absorption spectrum of the cobalt(II)-peptide complex was consistent with tetrahedral metal coordination by three sulfur and one imidazole nitrogen ligands. The peptide affinity for cobalt was less than for zinc, indicating metal specificity. Analysis of the fluorescence intensity pH profile, circular dichroism spectra, the effect of extrinsic quenchers indicated that at neutral pH (1) the free peptide up into a structure to place the tyrosine in an environment protected from solvent, (2) the peptide bound zinc via its three cysteines and one of its histidines resulting in little change to the polypeptide secondary structure or to the tyrosine solvent accessibility, and (3) when the peptide bound zinc, it bound directly to or caused the immobilization of the groups that had been intramolecularly collisionally quenching the tyrosine which resulted in the observed increases in tyrosine quantum yield and lifetime.

Human T-lymphotrophic virus type I nucleocapsid protein NCp15: structural study and stability of the N-terminal zinc-finger

“…This result is in accordance with the tetrahedral geometry described previously for the CCHC motif family [11,12]. Furthermore, in the UV region of the spectra, absorption bands at 313 and 352 nm appear, owing to a charge transfer from S V to Co# + , in accordance with published results on other peptides and proteins [29,30]. When performing spectroscopic studies at different pH values, we showed that metal co-ordination does not take place below pH 5.2, owing to histidine protonation.…”

“…The Zn# + co-ordination with these peptides was estimated to be 10$-10%-fold stronger than Co# + co-ordination (with K Co d and K Zn d approx. 1 µM and 100 pM respectively) [11,29,30]. In the present study we performed a detailed investigation of the stability of the NC N-terminal cobalt-finger and zinc-finger by using UV-visible absorption spectroscopy and HPLC chromatography.…”

Replisome-Mediated DNA Replication

“…This binding of DnaG/DnaB is critical for the recruitment of the primase to the replication fork. DnaG protein tightly binds one Zn, which is ligated by three cysteinyl sulfhydryls and one histidine nitrogen near the N terminus (72,73). Apoprimase, however, synthesizes primers in a sequence-specific manner, which eliminates a role for Zn in catalysis (73) and ascribes to it a structural one, possibly to prevent disulfide formation.…”

Oxidation Protection in Metal-Binding Peptide Motif and Its Application to Antibody for Site-Selective Conjugation

“…De novo designed metal binding motifs usually have tetra- or tri-peptide structures. Square planar or tetrahedral coordination environments can easily be achieved with tri- or tetra-peptide structures [ 25 , 26 ], providing a flexible and stable chelate ring for metal ions. Burrows et al prepared a CGH motif and characterized its coordination chemistry [ 27 ].…”