Dissociation of the DNA polymerase III holoenzyme β2 subunits is accompanied by conformational change at distal cysteines 333

M. A. Griep, C. S. McHenry

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Abstract

The β subunit of DNA polymerase III holoenzyme is in a dimer-monomer equilibrium at physiological β concentrations. Dissociation is accompanied by the fluorescence enhancement of a fluorophore attached to a unique sulfhydryl group of β (Griep, M.A., and McHenry, C.S. (1988) Biochemistry 27, 5210-5215). Sequencing of the isolated tryptic peptides of β revealed that the fluorescent maleimide group was attached to cysteine 333. The 2 residues, lysine 332 and glutamate 334, that flank this residue are hydrophilic and may place cysteine 333 on the surface of β, explaining its high reactivity. Fluorescence energy transfer permitted us to locate the uniquely labeled cysteines 333 of β at the distal ends of the β dimer. When the β dimer was dissociated to monomers, the accompanying alteration of the conformational state was reported by the fluorescein-5-maleimide (fluorescein)-labeled cysteines which were located far from the dimer interface. The carboxyl of fluorescein had a fluorescence pK(a) of 6.9 when β was in its dimeric state. The pK(a) decreased by 0.3 pH unit upon dissociation to monomers and resulted in the fluorescence enhancement that was observed when the signal was monitored at constant pH. The adjacent glutamate 334 apparently increased the pK(a) of the attached fluorescein when β was in its dimeric state. Movement of either the adjacent lysine 332 amino side chain to a closer position or glutamate 334 to a position further away could lower the pK(a) upon β monomerization. Thus, β undergoes a conformational change concomitant with dimer dissociation that was transmitted to the opposite ends of the β dimer. The pK(a) of fluorescein attached to the distal cysteines was shifted, leading to greater ionization and enhanced fluorescence.

Original languageEnglish (US)
Pages (from-to)20356-20363
Number of pages8
JournalJournal of Biological Chemistry
Volume265
Issue number33
Publication statusPublished - Dec 18 1990

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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