Conformational heterogeneity is revealed in the dissociation of the oligomeric chaperonin GroEL by high hydrostatic pressure.
We investigated the dissociation of tetradecameric GroEL by high hydrostatic pressure in the range of 1-2.5 kbar. Kinetics of the dissociation of GroEL in the absence and presence of Mg(2+) and/or KCl were monitored using light scattering. All of the kinetics were biphasic in nature. At any given pressure, only monomers and 14mers were produced, and below 2.5 kbar, the 14mers only partially dissociated to monomers, which did not significantly reassemble on depressurization. Under identical reaction conditions, the observed dissociation rates decreased by only 2-fold when the concentration of GroEL was increased by 20-fold. At 2.5 kbar the observed rates decreased exponentially with the increase in [KCl] and reached a minimum at approximately 75mM. Similarly, the rates decreased with the increase in [Mg(2+)] and reached a minimum at approximately 3 mM Mg(2+). In the presence of saturating amounts of Mg(2+) (10 mM) and KCl (100 mM), the rates were much faster than with 10 mM Mg(2+) alone. The results could be rationalized in terms of the presence of GroEL heterogeneity, which could not be assessed easily by common techniques such as sedimentation velocity, HPLC, gel electrophoresis, and dissociation by chaotropes. This heterogeneity is evidence of subpopulations of GroEL that dissociate at different pressures. At low pressures, the oligomer without added Mg(2+) only partially dissociates to monomers, leading to an apparent plateau in the kinetics, whereas in the presence of Mg(2+) the species are converted to a tighter Mg(2+)-bound species, leading to a much slower dissociation process. The presence of KCl in the sample also leads to similar heterogeneity.
Panda
Markandeswar
M
Department of Biochemistry, Mail Code 7760, University of Texas Health Science Center at San Antonio, 78229-3900, USA.
Horowitz
Paul M
PM
D018834:Chaperonin 60 / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry; D006874:Hydrostatic Pressure; D007700:Kinetics; D008024:Ligands; D008027:Light; D046911:Macromolecular Substances; D008274:Magnesium / Q000737:chemistry / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D012542:Scattering, Radiation
D018834:Chaperonin 60; D008024:Ligands; D046911:Macromolecular Substances; D011189:Potassium Chloride; D008274:Magnesium