Fluorescent Labeling of Cysteine 39 on Escherichia Coli Primase Places the Dye Near an Active Site

Mark A. Griep, Teresa N. Mesman

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Cysteine 39 of Escherichia coli primase is the most chemically reactive cysteine. Its high chemical reactivity is likely due to its proximity to primase's zinc, which is probably ligated to the adjacent residues 40-62. The zinc may stabilize the deprotonated form of cysteine 39 to make it chemically reactive. Primase is rapidly, site-specifically modified by fluorescein maleimiDe (FM) at this cysteine. Modification with FM at this residue does not lead to any activity loss in a coupled RNA/DNA synthesis assay, indicating that it is not a catalytically essential residue. In contrast, iodoacetamidefluorescein (IAF) modifies cysteine 39 more slowly and stoichiometrically inhibits activity. It was not clear why these two similar fluorescent dyes should have such different inhibitory effects when attached to the same cysteine. The IAF inhibition must be due to some property of the link between the fluorescein and the cysteine because that is how it differs from FM. The pKa's of the fluoresceins from both FMand IAF-modified primase are strongly shifted to lower values (approximately 5.4) compared to free fluorescein. These results strongly suggest that the deprotonated form of the fluoresceins are stabilized on primase by a strong interaction with the adjacent zinc in the zinc finger motif. The ability to place a noninhibitory FM at this site will be of great assistance in fluorescence energy transfer studies since the distances established to cysteine 39 will reflect the distance to the essential zinc finger motif.

Original languageEnglish (US)
Pages (from-to)673-682
Number of pages10
JournalBioconjugate Chemistry
Volume6
Issue number6
DOIs
StatePublished - Jan 1 1995

Fingerprint

DNA Primase
Labeling
Escherichia coli
Cysteine
Catalytic Domain
Zinc
Coloring Agents
Dyes
Fluoresceins
Zinc Fingers
Fluorescein
Chemical reactivity
RNA
Energy transfer
Assays
DNA
Fluorescence
Energy Transfer
Fluorescent Dyes
fluorescein 5-maleimide

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biomedical Engineering
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry

Cite this

Fluorescent Labeling of Cysteine 39 on Escherichia Coli Primase Places the Dye Near an Active Site. / Griep, Mark A.; Mesman, Teresa N.

In: Bioconjugate Chemistry, Vol. 6, No. 6, 01.01.1995, p. 673-682.

Research output: Contribution to journalArticle

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