Dissociation of the DNA polymerase III holoenzyme beta 2 subunits is accompanied by conformational change at distal cysteines 333.
The beta subunit of DNA polymerase III holoenzyme is in a dimer-monomer equilibrium at physiological beta concentrations. Dissociation is accompanied by the fluorescence enhancement of a fluorophore attached to a unique sulfhydryl group of beta (Griep, M. A., and McHenry, C. S. (1988) Biochemistry 27, 5210-5215). Sequencing of the isolated tryptic peptides of beta 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 beta, explaining its high reactivity. Fluorescence energy transfer permitted us to locate the uniquely labeled cysteines 333 of beta at the distal ends of the beta dimer. When the beta 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 pKa of 6.9 when beta was in its dimeric state. The pKa 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 pKa of the attached fluorescein when beta 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 pKa upon beta monomerization. Thus, beta undergoes a conformational change concomitant with dimer dissociation that was transmitted to the opposite ends of the beta dimer. The pKa of fluorescein attached to the distal cysteines was shifted, leading to greater ionization and enhanced fluorescence.
Griep
M A
MA
Department of Biochemistry, Biophysics and Genetics, University of Colorado Health Sciences Center, Denver 80262.
McHenry
C S
CS
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine; D004258:DNA Polymerase III / Q000302:isolation & purification / Q000378:metabolism*; D004735:Energy Transfer; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014357:Trypsin; D014364:Tryptophan
D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D014364:Tryptophan; D004258:DNA Polymerase III; D014357:Trypsin; D003545:Cysteine