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

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• 作者：Mark A. Griep ; Betsy J. Adkins ; Daniel Hromas ; Scott Johnson ; and Jennifer Miller
• 刊名：Biochemistry
• 出版年：1997
• 出版时间：January 21, 1997
• 年：1997
• 卷：36
• 期：3
• 页码：544 - 553
• 全文大小：263K
• 年卷期：v.36,no.3(January 21, 1997)
• ISSN：1520-4995

文摘

A 35-amino acid peptide corresponding to the putative "zincfinger" sequence of primase wasprepared to study its zinc binding properties. When zinc was addedto the peptide, it was found that thefluorescence quantum yield of the single tyrosine increased by 46% andthe average lifetime by 34%.The binding stoichiometry was one zinc per peptide. Below pH6.0 and above pH 8.5, the zinc-peptidebinding affinity was less than 1 µM and could be accuratelydetermined. Interpolation from those bindingconstants suggested that the affinity at pH 7.5 was between 10 and 100nM. The absorption spectrum ofthe cobalt(II)-peptide complex was consistent with tetrahedralmetal coordination by three sulfur andone imidazole nitrogen ligands. The peptide affinity for cobaltwas less than for zinc, indicating metalspecificity. Analysis of the fluorescence intensity pH profile,circular dichroism spectra, the effect ofextrinsic quenchers indicated that at neutral pH (1) the free peptidefolded up into a structure to place thetyrosine in an environment protected from solvent, (2) the peptidebound zinc via its three cysteines andone of its histidines resulting in little change to the polypeptidesecondary structure or to the tyrosinesolvent accessibility, and (3) when the peptide bound zinc, it bounddirectly to or caused the immobilizationof the groups that had been intramolecularly collisionally quenchingthe tyrosine which resulted in theobserved increases in tyrosine quantum yield and lifetime.