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.
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.
Griep
M A
MA
Department of Chemistry, University of Nebraska, Lincoln 68588-0304, USA.
Adkins
B J
BJ
Hromas
D
D
Johnson
S
S
Miller
J
J
D019915:DNA Primase; D004261:DNA Replication; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D014443:Tyrosine / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*; D016335:Zinc Fingers
D014443:Tyrosine; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D015032:Zinc